RSV F protein mutants

ABSTRACT

The present disclosure relates to RSV F protein mutants, nucleic acids or vectors encoding a RSV F protein mutant, compositions comprising a RSV F protein mutant or nucleic acid, and uses of the RSV F protein mutants, nucleic acids or vectors, and compositions.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/387,270 filed Dec. 23, 2015 and U.S. Provisional Application No.62/421,184 filed Nov. 11, 2016. The entire content of each of theforegoing applications is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“PC72226_02_SeqListing_ST25.txt” created on Nov. 21, 2016 and having asize of 1,286 KB. The sequence listing contained in this .txt file ispart of the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to vaccines in general and vaccinesagainst respiratory syncytial viruses specifically.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus, or RSV, is a respiratory virus that infectsthe lungs and breathing passages. RSV is the leading cause of seriousviral lower respiratory tract illness in infants worldwide and animportant cause of respiratory illness in the elderly. However, novaccines have been approved for preventing RSV infection.

RSV is a member of the Paramyxoviridae family. Its genome consists of asingle-stranded, negative-sense RNA molecule that encodes 11 proteins,including nine structural proteins (three glycoproteins and six internalproteins) and two non-structural proteins. The structural proteinsinclude three transmembrane surface glycoproteins: the attachmentprotein G, fusion protein F, and the small hydrophobic SH protein. Thereare two subtypes of RSV, A and B. They differ primarily in the Gglycoprotein, while the sequence of the F glycoprotein is more conservedbetween the two subtypes.

The mature F glycoprotein has three general domains: ectodomain (ED),transmembrane domain (TM), and a cytoplasmic tail (CT). CT contains asingle palmitoylated cysteine residue.

The F glycoprotein of human RSV is initially translated from the mRNA asa single 574-amino acid polypeptide precursor (referred to “F0” or “F0precursor”), which contains a signal peptide sequence (amino acids 1-25)at the N-terminus. Upon translation the signal peptide is removed by asignal peptidase in the endoplasmic reticulum. The remaining portion ofthe F0 precursor (i.e., residues 26-574) may be further cleaved at twopolybasic sites (a.a. 109/110 and 136/137) by cellular proteases (inparticular furin), removing a 27-amino acid intervening sequencedesignated pep27 (amino acids 110-136) and generating two linkedfragments designated F1 (C-terminal portion; amino acids 137-574) and F2(N-terminal portion; amino acids 26-109). F1 contains a hydrophobicfusion peptide at its N-terminus and two heptad-repeat regions (HRA andHRB). HRA is near the fusion peptide, and HRB is near the TM domain. TheF1 and F2 fragments are linked together through two disulfide bonds.Either the uncleaved F0 protein without the signal peptide sequence or aF1-F2 heterodimer can form a RSV F protomer. Three such protomersassemble to form the final RSV F protein complex, which is a homotrimerof the three protomers.

The F proteins of subtypes A and B are about 90 percent identical inamino acid sequence. An example sequence of the F0 precursor polypeptidefor the A subtype is provided in SEQ ID NO: 1 (A2 strain; GenBank GI:138251; Swiss Prot P03420), and for the B subtype is provided in SEQ IDNO: 2 (18537 strain; GenBank GI: 138250; Swiss Prot P13843). SEQ ID NO:1 and SEQ ID NO:2 are both 574 amino acid sequences. The signal peptidesequence for SEQ ID NO: 1 and SEQ ID NO:2 has also been reported asamino acids 1-25 (GenBank and UniProt). In both sequences the TM domainis from approximately amino acids 530 to 550, but has alternatively beenreported as 525-548. The cytoplasmic tail begins at either amino acid548 or 550 and ends at amino acid 574, with the palmitoylated cysteineresidue located at amino acid 550.

One of the primary antigens explored for RSV subunit vaccines is the Fprotein. The RSV F protein trimer mediates fusion between the virionmembrane and the host cellular membrane and also promotes the formationof syncytia. In the virion prior to fusion with the membrane of the hostcell, the largest population of F molecules forms a lollipop-shapedstructure, with the TM domain anchored in the viral envelope [Dormitzer,P. R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807,2012.]. This conformation is referred to as the pre-fusion conformation.Pre-fusion RSV F is recognized by monoclonal antibodies (mAbs) D25,AM22, and MPE8, without discrimination between oligomeric states.Pre-fusion F trimers are specifically recognized by mAb AM14 [Gilman MS, Moin S M, Mas V et al. Characterization of a prefusion-specificantibody that recognizes a quaternary, cleavage-dependent epitope on theRSV fusion glycoprotein. PLoS Pathogens, 11(7), 2015]. During RSV entryinto cells, the F protein rearranges from the pre-fusion state (whichmay be referred to herein as “pre-F”), through an intermediate extendedstructure, to a post-fusion state (“post-F”). During this rearrangement,the C-terminal coiled-coil of the pre-fusion molecule dissociates intoits three constituent strands, which then wrap around the globular headand join three additional helices to form the post-fusion six helixbundle. If a pre-fusion RSV F trimer is subjected to increasingly harshchemical or physical conditions, such as elevated temperature, itundergoes structural changes. Initially, there is loss of trimericstructure (at least locally within the molecule), and then rearrangementto the post-fusion form, and then denaturation of the domains.

To prevent viral entry, F-specific neutralizing antibodies presumablymust bind the pre-fusion conformation of F on the virion, or potentiallythe extended intermediate, before the viral envelope fuses with acellular membrane. Thus, the pre-fusion form of the F protein isconsidered the preferred conformation as the desired vaccine antigen[Ngwuta, J. O., Chen, M., Modjarrad, K., Joyce, M. G., Kanekiyo, M.,Kumar, A., Yassine, H. M., Moin, S. M., Killikelly, A. M., Chuang, G.Y., Druz, A., Georgiev, I. S., Rundlet, E. J., Sastry, M.,Stewart-Jones, G. B., Yang. Y., Zhang, B., Nason, M. C., Capella, C.,Peeples, M., Ledgerwood, J. E., Mclellan, J. S., Kwong, P. D., Graham,B. S., Science Translat. Med., 14, 7, 309 (2015)]. Upon extraction froma membrane with surfactants such as Triton X-100, Triton X-114, NP-40,Brij-35, Brij-58, Tween 20, Tween 80, Octyl glucoside, Octylthioglucoside, SDS, CHAPS, CHAPSO, or expression as an ectodomain,physical or chemical stress, or storage, the F glycoprotein readilyconverts to the post-fusion form [McLellan J S, Chen M, Leung S et al.Structure of RSV fusion glycoprotein trimer bound to apre-fusion-specific neutralizing antibody. Science 340, 1113-1117(2013); Chaiwatpongsakorn, S., Epand, R. F., Collins, P. L., Epand R.M., Peeples, M. E., J Virol. 85(8):3968-77 (2011); Yunus, A. S., JacksonT. P., Crisafi, K., Burimski, I., Kilgore, N. R., Zoumplis, D., Allaway,G. P., Wild, C. T., Salzwedel, K. Virology. 2010 Jan. 20;396(2):226-37]. Therefore, the preparation of pre-fusion F as a vaccineantigen has remained a challenge. Since the neutralizing and protectiveantibodies function by interfering with virus entry, it is postulatedthat an F antigen that elicits only post-fusion specific antibodies isnot expected to be as effective as an F antigen that elicits pre-fusionspecific antibodies. Therefore, it is considered more desirable toutilize an F vaccine that contains a F protein immunogen in thepre-fusion form (or potentially the extended intermediate form). Effortsto date have not yielded an RSV vaccine that has been demonstrated inthe clinic to elicit sufficient levels of protection to supportlicensure of an RSV vaccine. Therefore, there is a need for immunogensderived from a RSV F protein that have improved properties, such asenhanced immunogenicity or improved stability of the pre-fusion form, ascompared with the corresponding native RSV F protein, as well ascompositions comprising such an immunogen, such as a vaccine.

SUMMARY OF THE INVENTION

In some aspects, the present invention provides mutants of wild-type RSVF proteins, wherein the mutants display introduced mutations in theamino acid sequence relative to the amino acid sequence of thecorresponding wild-type RSV F protein and are immunogenic against thewild-type RSV F protein or against a virus comprising the wild-type Fprotein. The amino acid mutations in the mutants include amino acidsubstitutions, deletions, or additions relative to a wild-type RSV Fprotein.

In some embodiments, the present disclosure provides mutants of awild-type RSV F protein, wherein the introduced amino acid mutations aremutation of a pair of amino acid residues in a wild-type RSV F proteinto a pair of cysteines (“engineered disulfide mutation”). The introducedpair of cysteine residues allows for formation of a disulfide bondbetween the cysteine residues that stabilize the protein's conformationor oligomeric state, such as the pre-fusion conformation. Examples ofspecific pairs of such mutations include: 55C and 188C; 155C and 290C;103C and 148C; and 142C and 371C, such as S55C and L188C; S155C andS290C; T103C and I148C; and L142C and N371C.

In still other embodiments, the RSV F protein mutants comprise aminoacid mutations that are one or more cavity filling mutations. Examplesof amino acids that may be replaced with the goal of cavity fillinginclude small aliphatic (e.g. Gly, Ala, and Val) or small polar aminoacids (e.g. Ser and Thr) and amino acids that are buried in thepre-fusion conformation, but exposed to solvent in the post-fusionconformation. Examples of the replacement amino acids include largealiphatic amino acids (Ile, Leu and Met) or large aromatic amino acids(His, Phe, Tyr and Trp). In some specific embodiments, the RSV F proteinmutant comprises a cavity filling mutation selected from the groupconsisting of:

(1) substitution of S at position 55, 62, 155, 190, or 290 with I, Y, L,H, or NA;

(2) substitution of T at position 54, 58, 189, 219, or 397 with I, Y, L,H, or M;

(3) substitution of G at position 151 with A or H;

(4) substitution of A at position 147 or 298 with I, L, H, or M;

(5) substitution of V at position 164, 187, 192, 207, 220, 296, 300, or495 with I, Y, H; and

(6) substitution of R at position 106 with W.

In some particular embodiments, a RSV F protein mutant comprises atleast one cavity filling mutation selected from the group consisting of:T54H, S190I, and V296I.

In still other embodiments, the present disclosure provides RSV Fprotein mutants, wherein the mutants comprise electrostatic mutations,which decrease ionic repulsion or increase ionic attraction betweenresides in a protein that are proximate to each other in the foldedstructure. In several embodiments, the RSV F protein mutant includes anelectrostatic substitution that reduces repulsive ionic interactions orincreases attractive ionic interactions with acidic residues of Glu487and Asp489 from another protomer of RSV F trimer. In some specificembodiments, the RSV F protein mutant comprises an electrostaticmutation selected from the group consisting of:

(1) substitution of E at position 82, 92, or 487 by D, F, Q, T, S, L, orH;

(2) substitution of K at position 315, 394, or 399 by F, M, R, S, L, I,Q, or T;

(3) substitution of D at position 392, 486, or 489 by H, S, N, T, or P;and

(4) substitution of Rat position 106 or 339 by F, Q, N, or W.

In still other embodiments, the present disclosure provides RSV Fprotein mutants, which comprise a combination of two or more differenttypes of mutations selected from engineered disulfide mutations, cavityfilling mutations, and electrostatic mutations. In some particularembodiments, the present invention provides a mutant of a wild-type RSVF protein, which comprises a combination of mutations relative to thecorresponding wild-type RSV F protein, wherein the combination ofmutations is selected from the group consisting of:

(1) combination of T103C, I148C, S190I, and D486S;

(2) combination of T54H S55C L188C D486S;

(3) combination of T54H, T103C, I148C, S190I, V296I, and D486S;

(4) combination of T54H, S55C, L142C, L188C, V296I, and N371C;

(5) combination of S55C, L188C, and D486S;

(6) combination of T54H, S55C, L188C, and S190I;

(7) combination of S55C, L188C, S190I, and D486S;

(8) combination of T54H, S55C, L188C, S190I, and D486S;

(9) combination of S155C, S190I, S290C, and D486S;

(10) combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S,E487Q, and D489S; and

(11) combination of T54H, S155C, S190I, S290C, and V296I.

In another aspect, the present invention provides nucleic acid moleculesthat encode a RSV F protein mutant described herein. In some otherspecific embodiments, the present disclosure provides a nucleic acidmolecule encoding a RSV F protein mutant, wherein the nucleic acidmolecule comprises a nucleotide sequence selected from the groupconsisting of:

(1) a nucleotide sequence of SEQ ID NO:8;

(2) a nucleotide sequence of SEQ ID NO:9;

(3) a nucleotide sequence of SEQ ID NO:10;

(4) a nucleotide sequence of SEQ ID NO:11;

(5) a nucleotide sequence of SEQ ID NO:12;

(6) a nucleotide sequence of SEQ ID NO:13;

(7) a nucleotide sequence of SEQ ID NO:14;

(8) a nucleotide sequence of SEQ ID NO:15;

(9) a nucleotide sequence of SEQ ID NO:16;

(10) a nucleotide sequence of SEQ ID NO:17; and

(11) a nucleotide sequence of SEQ ID NO:18.

In another aspect, the invention provides compositions that comprise (1)a RSV F protein mutant described in the disclosure, or (2) a nucleicacid molecule or vector encoding such a RSV F protein mutant. In someparticular embodiments, the compositions are pharmaceuticalcompositions, which comprise a RSV F protein mutant provided by thepresent disclosure and a pharmaceutically acceptable carrier. In stillother particular embodiments, the pharmaceutical composition is avaccine.

The present disclosure also relates to use of a RSV F protein mutant,nucleic acids encoding such as a RSV F protein mutant, or vectors forexpressing such a RSV F protein mutant, or compositions comprising a RSVF protein mutant or nucleic acids. In some particular embodiments, thepresent disclosure provides a method of preventing RSV infection in asubject, comprising administering to the subject an effective amount ofa pharmaceutical composition, such as a vaccine, comprising a RSV Fprotein mutant, a nucleic acid encoding a RSV F protein mutant, or avector expressing a RSV F protein mutant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of the precursor polypeptidetemplate (SEQ ID NO:3) used for the construction of some of the RSV Fprotein mutants described in the Examples. The precursor polypeptideincludes a signal sequence (residues 1-25), F2 polypeptide (residues26-109), pep27 sequence (residues 110-136), F1 polypeptide (residues137-513), a T4 fibritin-derived trimerization domain (foldon; residues518-544), a thrombin recognition sequence (residues 547-552), ahistidine tag (residues 553-558), a Streptag II (561-568), and linkersequences (residues 514-517, 545-546, and 559-560). It also includesthree naturally occurring substitutions (P102A, I379V, and M447V)relative to the native RSV F sequence set forth in SEQ ID NO:1. Thefurin cleavage sites are shown as RARR and KKRKRR.

FIG. 2A depicts sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and western blot analysis of selectedpre-fusion F mutants (pXCS847, pXCS851 and pXCS852) under non-reducingconditions.

FIG. 2B shows sedimentation coefficient distributions of selectedmutants (pXCS847, pXCS851 and pXCS852) calculated from sedimentationvelocity experiments using an analytical ultracentrifuge.

FIGS. 3A and 3B depict the circular dichroism spectroscopy (CD) spectraof exemplary modified RSV F proteins with specific site mutations. Thefar-ultraviolet (UV) CD spectra of the designed mutants confirmsecondary structure integrity, and the near-UV CD spectra confirmtertiary structure integrity.

FIG. 4 depicts the time-dependent stress testing of purified DS-Cav1using two different monoclonal antibodies (mAbs) D25 and AM14 at twotemperatures (50° C. and 60° C.).

FIG. 5 depicts differential scanning calorimetry (DSC) experiments withpurified DS-Cav1 (Example 8). The experiments were done as described forthe designed pre-fusion F mutants. Solid line—initial DSC scan of thesample, dashed line—repeated scan of the same sample that was used inthe initial scan. The DSC peak largely recovers during the repeatedscan, indicating that conformational transition detected by the DSC isreversible.

FIG. 6A depicts far-UV CD spectra of DS-Cav1 stressed at 60° C. (Example8). CD spectra were recorded as described above for the designedpre-fusion RSV F mutants (Example 6). DS-Cav1 retains defined far-UV CDspectrum after up to 2 hours of incubation at 60° C., indicating that noglobal protein unfolding is taking place during that time.

FIG. 6B depicts near-UV CD spectra of DS-Cav1 stressed at 60° C.(Example 8). CD spectra were recorded as described above for thedesigned pre-fusion RSV F mutants (Example 6).

FIG. 7A depicts the protein concentration dependence of thermal stressresistance, as determined by the preservation of the pre-fusion Ftrimer-specific AM14 epitope (Example 8).

FIG. 7B depicts the protein concentration dependence of thermal stressresistance, as determined by the preservation of the pre-fusionF-specific D25 epitope (Example 8). Protein samples were seriallydiluted and subjected to the 50° C. stress for 1 hour. D25 reactivityremaining after the stress in relation to the control (unstressed)samples was assessed in ELISA assays.

FIG. 8 shows neutralizing antibody responses from mice immunized withDS-Cav1; wild-type F; or mutants pXCS852, pXCS855, pXCS830, pXCS853,pXCS780, pXCS898, pXCS851, pXCS874, pXCS881, pXCS738, or pXCS847; withor without aluminum phosphate as adjuvant. Results are reported as the50% geometric mean titer (GMT) from 10 mice per group. Each scatter plotreflects the response of individual mice with 10 animals total pergroup. The line within each group indicates the geometric mean 50%neutralizing antibody titer. “Wild-type F” refers to a wild-type Fectodomain recombinant construct.

FIGS. 9A and 9B describe correlations between the neutralizing antibodytiters elicited by and the stabilities of the engineered pre-fusion Fprotein mutants. Y-axis—neutralizing antibody titers elicited byimmunization of the mice with 0.25 μg antigen and no adjuvant or 0.025μg antigen with 0.1 mg/ml AlPO4 adjuvant. (Data are shown in Table 12.)X-axis—stability of the engineered mutants, as defined by the residualAM14 reactivity after thermal stress. (Data are shown in Table 8B.)

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to RSV F protein mutants, immunogeniccompositions comprising the RSV F protein mutants, methods for producingthe RSV F protein mutants, compositions comprising the RSV F proteinmutants, and nucleic acids that encode the RSV F protein mutants.

A. DEFINITIONS

The term “101F” refers to an antibody described in US 2006/0159695 A1,which has a heavy chain variable domain comprising an amino acidsequence of SEQ ID NO:30 and a light chain variable domain comprising anamino acid sequence of SEQ ID NO:31.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.”

The term “adjuvant” refers to a substance capable of enhancing,accelerating, or prolonging the body's immune response to an immunogenor immunogenic composition, such as a vaccine (although it is notimmunogenic by itself). An adjuvant may be included in the immunogeniccomposition, such as a vaccine, or may be administered separately fromthe immunogenic composition.

The term “administration” refers to the introduction of a substance orcomposition into a subject by a chosen route. Administration can belocal or systemic. For example, if the chosen route is intramuscular,the composition (such as a composition including a disclosed immunogen)is administered by introducing the composition into a muscle of thesubject.

The term “AM14” refers to an antibody described in WO 2008/147196 A2,which has a heavy chain variable domain comprising an amino acidsequence of SEQ ID NO:24 and a light chain variable domain comprising anamino acid sequence of SEQ ID NO:25.

The term “AM22” refers to an antibody described in WO 2011/043643 A1,which has a heavy chain variable domain comprising an amino acidsequence of SEQ ID NO:26 and a light chain variable domain comprising anamino acid sequence of SEQ ID NO:27.

The term “antigen” refers to a molecule that can be recognized by anantibody. Examples of antigens include polypeptides, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell.

The term “conservative substitution” refers to the substitution of anamino acid with a chemically similar amino acid. Conservative amino acidsubstitutions providing functionally similar amino acids are well knownin the art. The following six groups each contain amino acids that areconservative substitutions for one another:

1) alanine (A), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V); and

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The term “D25” refers to an antibody described in WO 2008/147196 A2,which has a heavy chain variable domain comprising an amino acidsequence of SEQ ID NO:22 and a light chain variable domain comprising anamino acid sequence of SEQ ID NO:23.

The term “degenerate variant” of a reference polynucleotide refers to apolynucleotide that differs in the nucleotide sequence from thereference polynucleotide but encodes the same polypeptide sequence asencoded by the reference polynucleotide. There are 20 natural aminoacids, most of which are specified by more than one codon. For instance,the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acidarginine. Thus, at every position where an arginine is specified withina protein encoding sequence, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given polypeptide.

The term “DS-Cav1” refers to the recombinanRSV F protein having theamino acid sequence described in McLellan, et al., Science, 342(6158),592-598, 2013. DS-Cav1 contains the following introduced amino acidsubstitutions: S155C, 5290C, S190F, and V207L.

The term “effective amount” refers to an amount of agent that issufficient to generate a desired response. For instance, this can be theamount necessary to inhibit viral replication or to measurably alteroutward symptoms of the viral infection.

The term “epitope” (or “antigenic determinant” or “antigenic site”)refers to the region of an antigen to which an antibody, B cellreceptor, or T cell receptor binds or responds. Epitopes can be formedfrom contiguous amino acids or noncontiguous amino acids juxtaposed bysecondary, tertiary, or quaternary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by higher order folding aretypically lost on treatment with denaturing solvents.

The term “F0 polypeptide” (F0) refers to the precursor polypeptide ofthe RSV F protein, which is composed of a signal polypeptide sequence, aF1 polypeptide sequence, a pep27 polypeptide sequence, and a F2polypeptide sequence. With rare exceptions the F0 polypeptides of theknown RSV strains consist of 574 amino acids.

The term “F1 polypeptide” (F1) refers to a polypeptide chain of a matureRSV F protein. Native F1 includes approximately residues 137-574 of theRSV F0 precursor and is composed of (from N- to C-terminus) anextracellular region (approximately residues 137-524), a transmembranedomain (approximately residues 525-550), and a cytoplasmic domain(approximately residues 551-574). As used herein, the term encompassesboth native F1 polypeptides and F1 polypeptides including modifications(e.g., amino acid substitutions, insertions, or deletion) from thenative sequence, for example, modifications designed to stabilize a Fmutant or to enhance the immunogenicity of a F mutant.

The term “F2 polypeptide” (F2) refers to the polypeptide chain of amature RSV F protein. Native F2 includes approximately residues 26-109of the RSV F0 precursor. As used herein, the term encompasses bothnative F2 polypeptides and F2 polypeptides including modifications(e.g., amino acid substitutions, insertions, or deletion) from thenative sequence, for example, modifications designed to stabilize a Fmutant or to enhance the immunogenicity of a F mutant. In native RSV Fprotein, the F2 polypeptide is linked to the F1 polypeptide by twodisulfide bonds to form a F2-F1 heterodimer. The term “foldon” or“foldon domain” refers to an amino acid sequence that is capable offorming trimers. One example of such foldon domains is the peptidesequence derived from bacteriophage T4 fibritin, which has the sequenceof GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:40).

The term “subject” refers to either a human or a non-human mammal. Theterm “mammal” refers to any animal species of the Mammalia class.Examples of mammals include: humans; non-human primates such as monkeys;laboratory animals such as rats, mice, guinea pigs; domestic animalssuch as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; andcaptive wild animals such as lions, tigers, elephants, and the like.

The term “glycoprotein” refers to a protein that containsoligosaccharide chains (glycans) covalently attached to polypeptideside-chains. The carbohydrate is attached to the protein in acotranslational or posttranslational modification known asglycosylation. The term “glycosylation site” refers to an amino acidsequence on the surface of a polypeptide, such as a protein, whichaccommodates the attachment of a glycan. An N-linked glycosylation siteis triplet sequence of NX(S/T) in which N is asparagine, X is anyresidue except proline, and (S/T) is a serine or threonine residue. Aglycan is a polysaccharide or oligosaccharide. Glycan may also be usedto refer to the carbohydrate portion of a glycoconjugate, such as aglycoprotein, glycolipid, or a proteoglycan.

The term “host cells” refers to cells in which a vector can bepropagated and its DNA or RNA expressed. The cell may be prokaryotic oreukaryotic.

The term “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence. Methods of alignment ofsequences for comparison are well known in the art. Once aligned, thenumber of matches is determined by counting the number of positionswhere an identical nucleotide or amino acid residue is present in bothsequences. The percent sequence identity is determined by dividing thenumber of matches either by the length of the sequence set forth in theidentified sequence, or by an articulated length (such as 100consecutive nucleotides or amino acid residues from a sequence set forthin an identified sequence), followed by multiplying the resulting valueby 100. For example, a peptide sequence that has 1166 matches whenaligned with a test sequence having 1554 amino acids is 75.0 percentidentical to the test sequence (1166÷1554*100=75.0).

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482, 1981, by the homology alignment algorithm of Needleman andWunsch, Mol. Biol. 48:443, 1970, by the search for similarity method ofPearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4th ed, Cold Spring Harbor, N.Y., 2012) and Ausubelet al. (In Current Protocols in Molecular Biology, John Wiley and Sons,New York, through supplement 104, 2013).

The term “immunogen” refers to a compound, composition, or substancethat is immunogenic as defined herein below.

The term “immunogenic” refers to the ability of a substance to cause,elicit, stimulate, or induce an immune response against a particularantigen, in a subject, whether in the presence or absence of anadjuvant.

The term “immune response” refers to any detectable response of a cellor cells of the immune system of a host mammal to a stimulus (such as animmunogen), including, but not limited to, innate immune responses(e.g., activation of Toll receptor signaling cascade), cell-mediatedimmune responses (e.g., responses mediated by T cells, such asantigen-specific T cells, and non-specific cells of the immune system),and humoral immune responses (e.g., responses mediated by B cells, suchas generation and secretion of antibodies into the plasma, lymph, and/ortissue fluids). Examples of immune responses include an alteration(e.g., increase) in Toll-like receptor activation, lymphokine (e.g.,cytokine (e.g., Th1, Th2 or Th17 type cytokines) or chemokine)expression or secretion, macrophage activation, dendritic cellactivation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cellactivation, B cell activation (e.g., antibody generation and/orsecretion), binding of an immunogen (e.g., antigen (e.g., immunogenicpolypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte(“CTL”) response, induction of a B cell response (e.g., antibodyproduction), and, expansion (e.g., growth of a population of cells) ofcells of the immune system (e.g., T cells and B cells), and increasedprocessing and presentation of antigen by antigen presenting cells. Theterm “immune response” also encompasses any detectable response to aparticular substance (such as an antigen or immunogen) by one or morecomponents of the immune system of a vertebrate animal in vitro.

The term ‘immunogenic composition” refers to a composition comprising animmunogen.

The term “MPE8” refers to an antibody described in Corti et al. [Corti,D., Bianchi, S., Vanzetta, F., Minola, A., Perez, L., Agatic, G.,Lanzavecchia, A. Cross-neutralization of four paramyxoviruses by a humanmonoclonal antibody. Nature, 501(7467), 439-443 (2013)], which has aheavy chain variable domain comprising an amino acid sequence of SEQ IDNO:28 and a light chain variable domain comprising an amino acidsequence of SEQ ID NO:29. The term “mutant” of a wild-type RSV Fprotein, “mutant” of a RSV F protein, “RSV F protein mutant,” or“modified RSV F protein” refers to a polypeptide that displaysintroduced mutations relative to a wild-type F protein and isimmunogenic against the wild-type F protein.

The term “mutation” refers to deletion, addition, or substitution ofamino acid residues in the amino acid sequence of a protein orpolypeptide as compared to the amino acid sequence of a referenceprotein or polypeptide. Throughout the specification and claims, thesubstitution of an amino acid at one particular location in the proteinsequence is referred to using a notation “(amino acid residue in wildtype protein)(amino acid position)(amino acid residue in engineeredprotein)”. For example, a notation Y75A refers to a substitution of atyrosine (Y) residue at the 75th position of the amino acid sequence ofthe reference protein by an alanine (A) residue (in a mutant of thereference protein). In cases where there is variation in the amino acidresidue at the same position among different wild-type sequences, theamino acid code preceding the position number may be omitted in thenotation, such as “75A.”

The term “native” or “wild-type” protein, sequence, or polypeptiderefers to a naturally existing protein, sequence, or polypeptide thathas not been artificially modified by selective mutations.

The term “pep27 polypeptide” or “pep27” refers to a 27-amino acidpolypeptide that is excised from the F0 precursor during maturation ofthe RSV F protein. The sequence of pep27 is flanked by two furincleavage sites that are cleaved by a cellular protease during F proteinmaturation to generate the F1 and F2 polypeptides.

The term “pharmaceutically acceptable carriers” refers to a material orcomposition which, when combined with an active ingredient, iscompatible with the active ingredient and does not cause toxic orotherwise unwanted reactions when administered to a subject,particularly a mammal. Examples of pharmaceutically acceptable carriersinclude solvents, surfactants, suspending agents, buffering agents,lubricating agents, emulsifiers, absorbants, dispersion media, coatings,and stabilizers.

The term “pre-fusion-specific antibody” refers to an antibody thatspecifically binds to the RSV F glycoprotein in a pre-fusionconformation, but does not bind to the RSV F protein in a post-fusionconformation. Exemplary pre-fusion-specific antibodies include the D25,AM22, 5C4, MPE8, and AM14 antibodies.

The term “pre-fusion trimer-specific antibody” refers to an antibodythat specifically binds to the RSV F glycoprotein in a pre-fusion,trimeric conformation, but does not bind to the RSV F protein in apost-fusion conformation or in a pre-fusion conformation that is notalso trimeric. An exemplary pre-fusion trimer-specific antibody is theAM14 antibody. “Pre-fusion trimer-specific antibodies” are a subset of“pre-fusion-specific antibodies.”

The term “prime-boost vaccination” refers to an immunotherapy regimenthat includes administration of a first immunogenic composition (theprimer vaccine) followed by administration of a second immunogeniccomposition (the booster vaccine) to a subject to induce an immuneresponse. The primer vaccine and the booster vaccine typically containthe same immunogen and are presented in the same or similar format.However, they may also be presented in different formats, for exampleone in the form of a vector and the other in the form of a naked DNAplasmid. The skilled artisan will understand a suitable time intervalbetween administration of the primer vaccine and the booster vaccine.Further, the primer vaccine, the booster vaccine, or both primer vaccineand the booster vaccine additionally include an adjuvant.

The term “pre-fusion conformation” refers to a structural conformationadopted by an RSV F protein or mutant that can be specifically bound by(i) antibody D25 when the RSV F protein or mutant is in the form of amonomer or trimer, or (ii) by antibody AM14 when the RSV F proteinmutant is in the form of a trimer. The pre-fusion trimer conformation isa subset of pre-fusion conformations.

The term “post-fusion conformation” refers to a structural conformationadopted by the RSV F protein that is not specifically bound by D25,AM22, or AM14. Native F protein adopts the post-fusion conformationsubsequent to the fusion of the virus envelope with the host cellularmembrane. RSV F protein may also assume the post-fusion conformationoutside the context of a fusion event, for example, under stressconditions such as heat and low osmolality, when extracted from amembrane, when expressed as an ectodomain, or upon storage.

The term “soluble protein” refers to a protein capable of dissolving inaqueous liquid and remaining dissolved. The solubility of a protein maychange depending on the concentration of the protein in the water-basedliquid, the buffering condition of the liquid, the concentration ofother solutes in the liquid, for example salt and proteinconcentrations, and the temperature of the liquid.

The term “specifically bind,” in the context of the binding of anantibody to a given target molecule, refers to the binding of theantibody with the target molecule with higher affinity than its bindingwith other tested substances. For example, an antibody that specificallybinds to the RSV F protein in pre-fusion conformation is an antibodythat binds RSV F protein in pre-fusion conformation with higher affinitythan it binds to the RSV F protein in the post-fusion conformation.

The term “therapeutically effective amount” refers to the amount ofagent that is sufficient to prevent, treat (including prophylaxis),reduce and/or ameliorate the symptoms and/or underlying causes of adisorder.

The term “vaccine” refers to a pharmaceutical composition comprising animmunogen that is capable of eliciting a prophylactic or therapeuticimmune response in a subject. Typically, a vaccine elicits anantigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen.

The term “vector” refers to a nucleic acid molecule capable oftransporting or transferring a foreign nucleic acid molecule. The termencompasses both expression vectors and transcription vectors. The term“expression vector” refers to a vector capable of expressing the insertin the target cell, and generally contains control sequences, such asenhancer, promoter, and terminator sequences, that drive expression ofthe insert. The term “transcription vector” refers to a vector capableof being transcribed but not translated. Transcription vectors are usedto amplify their insert. The foreign nucleic acid molecule is referredto as “insert” or “transgene.” A vector generally consists of an insertand a larger sequence that serves as the backbone of the vector. Basedon the structure or origin of vectors, major types of vectors includeplasmid vectors, cosmid vectors, phage vectors such as lambda phage,viral vectors such as adenovirus (Ad) vectors, and artificialchromosomes.

B. RSV F PROTEIN MUTANTS

In some aspects, the present invention provides mutants of wild-type RSVF proteins, wherein the mutants display introduced mutations in theamino acid sequence relative to the amino acid sequence of thecorresponding wild-type RSV F protein and are immunogenic against thewild-type RSV F protein or against a virus comprising the wild-type Fprotein. In certain embodiments, the RSV F mutants possess certainbeneficial characteristics, such as increased immunogenic properties orimproved stability in the pre-fusion conformation of the mutants orpre-fusion trimeric conformation of the mutant, as compared to thecorresponding wild-type F protein. In still other embodiments, thepresent disclosure provide RSV F mutants that display one or moreintroduced mutations as described herein and bind to a pre-fusionspecific antibody selected from antibody D25 or antibody AM14.

The introduced amino acid mutations in the RSV F protein mutants includeamino acid substitutions, deletions, or additions. In some embodiments,the only mutations in the amino acid sequence of the mutants are aminoacid substitutions relative to a wild-type RSV F protein.

The amino acid sequence of a large number of native RSV F proteins fromdifferent RSV subtypes, as well as nucleic acid sequences encoding suchproteins, is known in the art. For example, the sequence of severalsubtype A, B and bovine RSV F0 precursor proteins are set forth in SEQID NOs:1, 2, 4, 6 and 81-270.

The native RSV F protein exhibits remarkable sequence conservationacross RSV subtypes. For example, RSV subtypes A and B share 90%sequence identity, and RSV subtypes A and B each share 81% sequenceidentify with bovine RSV F protein, across the F0 precursor molecule.Within RSV subtypes the F0 sequence identity is even greater; forexample within each of RSV A, B, and bovine subtypes, the RSV F0precursor protein has about 98% sequence identity. Nearly all identifiedRSV F0 precursor sequences consist of 574 amino acids in length, withminor differences in length typically due to the length of theC-terminal cytoplasmic tail. Sequence identity across various native RSVF proteins is known in the art (see, for example, WO2014/160463). Tofurther illustrate the level of the sequence conservation of F proteins,non-consensus amino acid residues among F0 precursor polypeptidesequences from representative RSV A strains and RSV B strains areprovided in Tables A and B, respectively (where non-consensus aminoacids were identified following alignment of selected F proteinsequences from RSV A strains with ClustaIX (v. 2)).

In view of the substantial conservation of RSV F sequences, a person ofordinary skill in the art can easily compare amino acid positionsbetween different native RSV F sequences to identify corresponding RSV Famino acid positions between different RSV strains and subtypes. Forexample, across nearly all identified native RSV F0 precursor proteins,the furin cleavage sites fall in the same amino acid positions. Thus,the conservation of native RSV F protein sequences across strains andsubtypes allows use of a reference RSV F sequence for comparison ofamino acids at particular positions in the RSV F protein. For thepurposes of this disclosure (unless context indicates otherwise), theRSV F protein amino acid positions are given with reference to thesequence of the F0 precursor polypeptide set forth in SEQ ID NO: 1 (theamino acid sequence of the full length native F precursor polypeptide ofthe RSV A2 strain; corresponding to GenInfo Identifier GI 138251 andSwiss Prot identifier P03420). However, it should be noted, and one ofskill in the art will understand, that different RSV F0 sequences mayhave different numbering systems, for example, if there are additionalamino acid residues added or removed as compared to SEQ ID NO:1. Assuch, it is to be understood that when specific amino acid residues arereferred to by their number, the description is not limited to onlyamino acids located at precisely that numbered position when countingfrom the beginning of a given amino acid sequence, but rather that theequivalent/corresponding amino acid residue in any and all RSV Fsequences is intended even if that residue is not at the same precisenumbered position, for example if the RSV sequence is shorter or longerthan SEQ ID NO:1, or has insertions or deletions as compared to SEQ IDNO: 1.

B-1. Structure of the RSV F Protein Mutants

The RSV F protein mutants provided by the present disclosure comprise aF1 polypeptide and a F2 polypeptide. In several embodiments, the mutantsfurther comprise a trimerization domain. In some embodiments, either theF1 polypeptide or the F2 polypeptide includes at least one introducedmodification (e.g., amino acid substitution) as described in detailherein below. In some other embodiments, each of the F1 polypeptide andF2 polypeptide includes at least one introduced modification (e.g.,amino acid substitution) as described in detail herein below.

B-1(a). F1 Polypeptide and F2 Polypeptide of the RSV F Mutants

In some embodiments, the mutants are in the mature form of the RSV Fprotein, which comprises two separate polypeptide chains, namely the F1polypeptide and F2 polypeptide. In some other embodiments, the F2polypeptide is linked to the F1 polypeptide by one or two disulfidebonds to form a F2-F1 polypeptide heterodimer. In still otherembodiments, the RSV F mutants are in the form a single chain protein,wherein the F2 polypeptide is linked to the F1 polypeptide by a peptidebond or peptide linker. Any suitable peptide linkers for joining twopolypeptide chains together may be used. Examples of such linkersinclude G, GG, GGG, GS, and SAIG linker sequences. The linker may alsobe the full length pep27 sequence or a fragment thereof.

The F1 polypeptide chain of the mutant may be of the same length as thefull length F1 polypeptide of the corresponding wild-type RSV F protein;however, it may also have deletions, such as deletions of 1 up to 60amino acid residues from the C-terminus of the full-length F1polypeptide. A full-length F1 polypeptide of the RSV F mutantscorresponds to amino acid positions 137-574 of the native RSV F0precursor, and includes (from N- to C-terminus) an extracellular region(residues 137-524), a transmembrane domain (residues 525-550), and acytoplasmic domain (residues 551-574). It should be noted that aminoacid residues 514 onwards in a native F1 polypeptide sequence areoptional sequences in a F1 polypeptide of the RSV F mutants providedherein, and therefore may be absent from the F1 polypeptide of themutant.

In some embodiments, the F1 polypeptide of the RSV F mutants lacks theentire cytoplasmic domain. In other embodiments, the F1 polypeptidelacks the cytoplasmic domain and a portion of or all entiretransmembrane domain. In some specific embodiments, the mutant comprisesa F1 polypeptide wherein the amino acid residues from position 510, 511,512, 513, 514, 515, 520, 525, or 530 through 574 are absent. Typically,for mutants that are linked to trimerization domain, such as a foldon,amino acids 514 through 754 can be absent. Thus, in some specificembodiment, amino acid residues 514 through 574 are absent from the F1polypeptide of the mutant. In still other specific embodiments, the F1polypeptide of the RSV F mutants comprises or consists of amino acidresidues 137-513 of a native F0 polypeptide sequence, such as any of theF0 precursor sequence set forth in SEQ ID Nos: 1, 2, 4, 6, and 81-270.

On the other hand, the F1 polypeptide of the RSV F mutant may include aC-terminal linkage to a trimerization domain, such as a foldon. Many ofthe sequences of the RSV F mutants disclosed herein include a sequenceof protease cleavage site, such as thrombin cleavage site (LVPRGS),protein tags, such as 6×His-tag (HHHHHH) and Streptag II (WSHPGFEK), orlinker sequences (such as GG and GS) (See FIG. 1) that are not essentialfor the function of the RSV F protein, such as for induction of animmune response. A person skilled in the art will recognize suchsequences, and when appropriate, understand that these sequences are notincluded in a disclosed RSV F mutant.

In the RSV F mutants provided by the present disclosure, the F2polypeptide chain may be of the same length as the full-length F2polypeptide of the corresponding wild-type RSV F protein; it may alsohave deletions, such as deletions of 1, 2, 3, 4, 5, 6, 7, or 8 aminoacid residues from the N-terminus or C-terminus of the F2 polypeptide.

The mutant in F0 form (i.e., a single chain polypeptide comprising theF2 polypeptide joined to the F1 polypeptide with or without partial orfull length pep 27) or F1-F2 heterodimer form may form a protomer. Themutant may also be in the form of a trimer, which comprises three of thesame protomer. Further, the mutants may be glycosylated proteins (i.e.,glycoproteins) or non-glycosylated proteins. The mutant in F0 form mayinclude, or may lack, the signal peptide sequence.

The F1 polypeptide and F2 polypeptide of the RSV F protein mutants towhich one or more mutations are introduced can be from any wild-type RSVF proteins known in the art or discovered in the future, including,without limitations, the F protein amino acid sequence of RSV subtype A,and subtype B strains, including A2 Ontario and Buenos Aires, or anyother subtype. In some embodiments, the RSV F mutant comprises a F1and/or a F2 polypeptide from a RSV A virus, for example, a F1 and/or F2polypeptide from a RSV F0 precursor protein set forth in any one of SEQID NOs: 1, 2, 4, 6, and 81-270 to which one or more mutations areintroduced. In some other embodiments, the RSV F mutant comprises a F1and/or a F2 polypeptide from a RSV B virus, for example, a F1 and/or F2polypeptide from a RSV F0 precursor protein set forth in any one of SEQID NOs:2, and 211-263 to which one or more mutations are introduced. Instill other embodiments, the RSV F mutant comprises a F1 and/or a F2polypeptide from a RSV bovine virus, for example, a F1 and/or F2polypeptide from a RSV F0 precursor protein set forth in any one of SEQID NOs:264-270 to which one or more mutations are introduced.

In some embodiments, the RSV F protein mutants comprise aF1-polypeptide, a F2 polypeptide, and one or more introduced amino acidmutations as described herein below, wherein the F1 polypeptidecomprises 350 consecutive amino acids and is at least 90, 95, 98, or 99percent identical to amino acids 137-513 of any of the sequences of SEQID NO:1, 4, and 81-210, wherein the F2 polypeptide comprises 70consecutive amino acids and is at least 90, 95, 98, or 99 percentidentical to amino acids 26-109 of any of the sequence of SEQ ID NO:1,4, and 81-210 and wherein RSV F protein mutant is stabilized inpre-fusion conformation, whether as monomer or trimer. In someembodiments, the F1 polypeptide comprises 350 consecutive amino acidsand is at least 90, 95, 98, or 99 percent identical to amino acids137-513 of any of the sequence of SEQ ID NOs:2, 6, and 211-263, and theF2 polypeptide comprises 70 consecutive amino acids and is at least 90,95, 98, or 99 percent identical to amino acids 26-109 of any of thesequence of SEQ ID NOs:2, 6, and 211-263. In some other embodiments, theRSV F protein mutant is stabilized in pre-fusion trimer conformation.

B-1(b) Trimerization Domains

In several embodiments, the RSV F mutant provided by the presentdisclosure is linked to a trimerization domain. In some embodiments, thetrimerization domain promotes the formation of trimer of three F1/F2heterodimers.

Several exogenous multimerization domains that promote formation ofstable trimers of soluble proteins are known in the art. Examples ofsuch multimerization domains that can be linked to a mutant provided bythe present disclosure include: (1) the GCN4 leucine zipper (Harbury etal. 1993 Science 262: 1401-1407); (2) the trimerization motif from thelung surfactant protein (Hoppe et al. 1994 FEB S Lett 344: 191-195); (3)collagen (McAlinden et al. 2003 Biol Chem 278:42200-42207); and (4) thephage T4 fibritin foldon (Miroshnikov et al. 1998 Protein Eng11:329-414). In some embodiments, a foldon domain is linked to a Fmutant at the C-terminus of F1 polypeptide. In specific embodiments, thefoldon domain is a T4 fibritin foldon domain, such as the amino acidsequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 40).

Typically, the multimerization domain is positioned C-terminal to the F1polypeptide. It may join directly to the F1 polypeptide chain.Optionally, the multimerization domain is connected to the F1polypeptide via a linker, such as an amino acid linker, for example thesequence GG, GS, or SAIG. The linker can also be a longer linker (forexample, including the repeat sequence GG). Numerous conformationallyneutral linkers are known in the art that can be used in the mutantsprovided by the present disclosure. In some embodiments, the F mutantcomprising a foldon domain include a protease cleavage site for removingthe foldon domain from the F1 polypeptide, such as a thrombin sitebetween the F1 polypeptide and the foldon domain.

B-2. Introduced Mutations in the RSV F Protein Mutants

The RSV F mutants provided by the present disclosure comprise a F1polypeptide and a F2 polypeptide, wherein (1) either the F1 polypeptideor (2) the F2 polypeptide, or (3) both the F1 polypeptide and F2polypeptide include one or more introduced amino acid mutations relativeto the amino acid sequence of the corresponding native F protein. Theintroduction of such amino acid mutations in the RSV F mutants mayconfer a beneficial property to the mutants, such as enhancedimmunogenicity, improved stability, or formation or improved stabilityof certain desired physical form or conformation of the mutants. Suchintroduced amino acid mutations are referred to as “engineered disulfidebond mutations,” “cavity filling mutations,” or “electrostaticmutations,” and are described in detail herein below. RSV F mutants thatinclude any additional mutations are also encompassed by the inventionso long as the immunogenic property of the mutants is not substantiallyadversely affected by the additional mutations.

B-2(a) Engineered Disulfide Bond Mutations

In some embodiments, RSV F mutants provided by the present disclosureinclude one or more engineered disulfide bond mutations. The term“engineered disulfide bond mutation” or “engineered disulfide mutation”refers to mutation of a pair of amino acid residues in a wild-type RSV Fprotein to a pair of cysteine residues. The introduced pair of cysteineresidues allows for formation of a disulfide bond between the introducedcysteine residues, which disulfide bond serves to stabilize theprotein's conformation or oligomeric state, such as pre-fusionconformation. For stabilizing the pre-fusion conformation of the mutant,the residue pairs for mutation to cysteine should be in close proximityin the pre-fusion conformation but distant in the post-fusionconformation. Such residues can be identified by suitable methods knownin the art, such as by visual inspection of a crystal structure of RSV Fin a pre-fusion conformation, or more quantitative selection usingcomputational protein design software (such as BioLuminate™[BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™[Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™[Molecular Operating Environment, Chemical Computing Group Inc.,Montreal, 2015], and Rosetta™ [Rosetta, University of Washington,Seattle, 2015]). Preferably, the distance between the pair of residues(e.g. the beta carbons) is less than 8 Å in a pre-fusion conformation,but more than 20 Å in a post-fusion conformation.

In some embodiments, the RSV F protein mutants comprise only oneengineered disulfide mutation (“single engineered disulfide mutation”).In some other embodiments, the RSV F protein mutants comprise at leasttwo engineered disulfide mutations, wherein each pair of the cysteineresidues of the engineered disulfide mutations are appropriatelypositioned when RSV F protein mutant is in pre-fusion conformation(“double engineered disulfide mutation”).

In some specific embodiments, the present disclosure provides a RSV Fmutant comprising at least one engineered disulfide bond mutation,wherein the mutant comprises the same introduced mutations that are inany of the exemplary mutants provided in Tables 1 and 4-6. The exemplaryRSV F mutants provided in Tables 1 and 4-6 are based on the same nativeF0 sequence of RSV A2 strain with three naturally-occurringsubstitutions at positions 102, 379, and 447 (SEQ ID NO:3). The sameintroduced mutations in each of the mutants can be made to a native F0polypeptide sequence of any other RSV subtype or strain to arrive atdifferent RSV F mutants, such as a native F0 polypeptide sequence setforth in any of the SEQ ID NOs: 1, 2, 4, 6, and 81-270. RSV F mutantsthat are based on a native F0 polypeptide sequence of any other RSVsubtype or strain and comprise any of the engineered disulfide mutationsare also within the scope of the invention. In some particularembodiments, a RSV F protein mutant comprises at least one engineereddisulfide mutation selected from the group consisting of: 55C and 188C;155C and 290C; 103C and 148C; and 142C and 371C, such as S55C and L188C,S155C and S290C, T103C and I148C, or L142C and N371C.

In some embodiments, the present disclosure provides RSV F proteinmutants, wherein the amino acid mutations are mutation of a pair ofamino acid residues in the HRB region (approximately amino acids476-524) of a RSV F protein to a pair of cysteines. The introduced pairof cysteine residues allows for formation of a disulfide bond betweenthe cysteine residues from two adjacent F2-F1 mutant protomers of atrimer. The disulfide linking two protomers in a trimer serves tostabilize the mutant in a trimeric state. Examples of specific pairs ofsuch mutations include: 508C and 509C; 515C and 516C; 522C and 523C,such as K508C and S509C, N515C and V516C, or T522C and T523C. In someembodiments, the RSV F mutants comprise (1) at least one pair ofcysteine mutations in the HRB region and (2) at least one introducedmutation outside of the HRB region selected from an engineered disulfidebond mutation as described herein above, a cavity filling mutation asdescribed herein below, an electrostatic mutation as described hereinbelow, or a combination of any of these mutations.

B-2(b) Cavity Filling Mutations.

In other embodiments, the present disclosure provides RSV F mutants thatcomprise one or more cavity filling mutations. The term “cavity fillingmutation” refers to the substitution of an amino acid residue in thewild-type RSV F protein by an amino acid that is expected to fill aninternal cavity of the mature RSV F protein. In one application, suchcavity-filling mutations contribute to stabilizing the pre-fusionconformation of a RSV F protein mutant. The cavities in the pre-fusionconformation of the RSV F protein can be identified by methods known inthe art, such as by visual inspection of a crystal structure of RSV F ina pre-fusion conformation, or by using computational protein designsoftware (such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York,2015], Discovery Studio™ [Discovery Studio Modeling Environment,Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment,Chemical Computing Group Inc., Montreal, 2015], and Rosetta™ [Rosetta,University of Washington, Seattle, 2015]). The amino acids to bereplaced for cavity-filling mutations typically include small aliphatic(e.g. Gly, Ala, and Val) or small polar amino acids (e.g. Ser and Thr).They may also include amino acids that are buried in the pre-fusionconformation, but exposed to solvent in the post-conformation. Thereplacement amino acids can be large aliphatic amino acids (Ile, Leu andMet) or large aromatic amino acids (His, Phe, Tyr and Trp). For example,in several embodiments, the RSV F protein mutant includes a T54Hmutation.

In some specific embodiments, the present disclosure provides a RSV Fprotein mutant that comprises one or more cavity filling mutationsselected from the group consisting of:

1) substitution of the amino acid at position 55, 62, 155, 190, or 290with I, Y, L, H, or NA;

2) substitution of the amino acid at position 54, 58, 189, 219, or 397with I, Y, L, H, or M;

3) substitution of the amino acid at position 151 with A or H;

4) substitution of the amino acid at position 147 or 298 with I, L, H,or M;

5) substitution of the amino acid at position 164, 187, 192, 207, 220,296, 300, or 495 with I, Y, H; and

6) substitution of the amino acid at position 106 with W.

In some further specific embodiments, the RSV F protein mutant comprisesone or more cavity filling mutations selected from the group consistingof:

1) substitution of Sat position 55, 62, 155, 190, or 290 with I, Y, L,H, or M;

2) substitution of T at position 54, 58, 189, 219, or 397 with I, Y, L,H, or M;

3) substitution of G at position 151 with A or H;

4) substitution of A at position 147 or 298 with I, L, H, or M;

5) substitution of V at position 164, 187, 192, 207, 220, 296, 300, or495 with I, Y, H; and

6) substitution of R at position 106 with W.

In some specific embodiments, the present disclosure provides a RSV Fmutant comprising one or more cavity filling mutations, wherein themutant comprises the cavity filling mutations in any of the mutantsprovided in Tables 2, 4, and 6. RSV F mutants provided in Tables 2, 4,and 6 are based on the same native F0 sequence of RSV A2 strain withthree naturally occurring substitutions at positions 102, 379, and 447(SEQ ID NO:3). The same introduced mutations in each of the mutants canbe made to a native F0 polypeptide sequence of any other RSV subtype orstrain to arrive at different RSV F mutants, such as a native F0polypeptide sequence set forth in any of the SEQ ID NOs:1, 2, 4, 6, and81-270. The RSV F mutants that are based on a native F0 polypeptidesequence of any other RSV subtype or strain and comprise any of the oneor more cavity filling mutations are also within the scope of theinvention. In some particular embodiments, a RSV F protein mutantprovided by the present disclosure comprises at least one cavity fillingmutation selected from the group consisting of: T54H, S190I, and V296I.

B-2 (c) Electrostatic Mutations.

In still other embodiments, the present disclosure provides RSV Fprotein mutants that include one or more electrostatic mutations. Theterm “electrostatic mutation” refers to an amino acid mutationintroduced to a wild-type RSV F protein that decreases ionic repulsionor increase ionic attraction between residues in a protein that areproximate to each other in the folded structure. As hydrogen bonding isa special case of ionic attraction, electrostatic mutations may increasehydrogen bonding between such proximate residues. In one example, anelectrostatic mutation may be introduced to improve trimer stability. Insome embodiments, an electrostatic mutation is introduced to decreaserepulsive ionic interactions or increase attractive ionic interactions(potentially including hydrogen bonds) between residues that are inclose proximity in the RSV F glycoprotein in its pre-fusion conformationbut not in its post-fusion conformation. For example, in the pre-fusionconformation, the acidic side chain of Asp486 from one protomer of theRSV F glycoprotein trimer is located at the trimer interface andstructurally sandwiched between two other acidic side chains of Glu487and Asp489 from another protomer. On the other hand, in the post-fusionconformation, the acidic side chain of Asp486 is located on the trimersurface and exposed to solvent. In several embodiments, the RSV Fprotein mutant includes an electrostatic D486S substitution that reducesrepulsive ionic interactions or increases attractive ionic interactionswith acidic residues of Glu487 and Asp489 from another protomer of RSV Ftrimer. Introduction of an electrostatic mutation may increase themelting temperature (Tm) of the pre-fusion conformation or pre-fusiontrimer conformation of the RSV F protein.

Unfavorable electrostatic interactions in a pre-fusion or pre-fusiontrimer conformation can be identified by method known in the art, suchas by visual inspection of a crystal structure of RSV F in a pre-fusionor pre-fusion trimer conformation, or by using computational proteindesign software (such as BioLuminate™ [BioLuminate, Schrodinger LLC, NewYork, 2015], Discovery Studio™ [Discovery Studio Modeling Environment,Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment,Chemical Computing Group Inc., Montreal, 2015.], and Rosetta™ [Rosetta,University of Washington, Seattle, 2015.])

In some specific embodiments, the RSV F protein mutant provided by thepresent disclosure comprises at least one electrostatic mutationselected from the group consisting of:

1) substitution of the amino acid at position 82, 92, or 487 by D, F, Q,T, S, L, or H;

2) substitution of the amino acid at position 315, 394, or 399 by F, M,R, S, L, I, Q, or T;

3) substitution of the amino acid at position 392, 486, or 489 by H, S,N, T, or P; and

4) substitution of the amino acid at position 106 or 339 by F, Q, N, orW.

In some further specific embodiments, the RSV F protein mutant comprisesat least one electrostatic mutation selected from the group consistingof:

1) substitution of E at position 82, 92, or 487 by D, F, Q, T, S, L, orH;

2) substitution of K at position 315, 394, or 399 by F, M, R, S, L, I,Q, or T;

3) substitution of D at position 392, 486, or 489 by H, S, N, T, or P;and

4) substitution of R at position 106 or 339 by F, Q, N, or W.

In some specific embodiments, the present disclosure provides a RSV Fmutant comprising one or more electrostatic mutations, wherein themutant comprises the electrostatic mutations in any of the mutantsprovided in Tables 3, 5, and 6. RSV F mutants provided in Tables 3, 5,and 6 are based on the same native F0 sequence of RSV A2 strain withthree naturally occurring substitutions at positions 102, 379, and 447(SEQ ID NO:3). The same introduced mutations in each of the mutants canbe made to a native F0 polypeptide sequence of any other RSV subtype orstrain to arrive at different RSV F mutants, such as a native F0polypeptide sequence set forth in any of the SEQ ID NOs:1, 2, 4, 6, and81-270. RSV F mutants that are based on a native F0 polypeptide sequenceof any other RSV subtype or strain and comprise any of the one or moreelectrostatic mutations are also within the scope of the invention. Insome particular embodiments, the RSV F protein mutant comprises mutationD486S.

B-2 (d) Combination of Engineered Disulfide Bond Mutations, CavityFilling Mutations, and Electrostatic Mutations.

In another aspect, the present disclosure provides RSV F proteinmutants, which comprise a combination of two or more different types ofmutations selected from engineered disulfide bond mutations, cavityfilling mutations, and electrostatic mutations, each as described hereinabove.

In some embodiments, the mutants comprise at least one engineereddisulfide bond mutation and at least one cavity filling mutation. Insome specific embodiments, the RSV F mutants include a combination ofmutations as noted in Table 4.

In some further embodiments, the RSV F protein mutants comprise at leastone engineered disulfide mutation and at least one electrostaticmutation. In some specific embodiments, the RSV F mutants include acombination of mutations as noted in Table 5.

In still other embodiments, the RSV F protein mutants comprise at leastone engineered disulfide mutation, at least one cavity filling mutation,and at least one electrostatic mutation. In some specific embodiments,the RSV F mutants include a combination of mutations as provided inTable 6.

In some particular embodiments, the present invention provides a RSV Fmutant that comprises a combination of mutations selected from the groupconsisting of:

(1) combination of 103C, 148C, 190I, and 486S;

(2) combination of 54H, 55C, 188C, and 486S;

(3) combination of 54H, 103C, 148C, 190I, 296I, and 486S;

(4) combination of 54H, 55C, 142C, 188C, 296I, and 371C;

(5) combination of 55C, 188C, and 486S;

(6) combination of 54H, 55C, 188C, and 190I;

(7) combination of 55C, 188C, 190I, and 486S;

(8) combination of 54H, 55C, 188C, 190I, and 486S;

(9) combination of 155C, 190I, 290C, and 486S;

(10) combination of 54H, 55C, 142C, 188C, 296I, 371C, 486S, 487Q, and489S; and

(11) combination of 54H, 155C, 190I, 290C, and 296I.

In some particular embodiments, the present invention provides a RSV Fmutant that comprises a combination of mutations selected from the groupconsisting of:

(1) combination of T103C, I148C, S190I, and D486S;

(2) combination of T54H, S55C, L188C, and D486S;

(3) combination of T54H, T103C, I148C, S190I, V296I, and D486S;

(4) combination of T54H, S55C, L142C, L188C, V296I, and N371C;

(5) combination of S55C, L188C, and D486S;

(6) combination of T54H, S55C, L188C, and S190I;

(7) combination of S55C, L188C, S190I, and D486S;

(8) combination of T54H, S55C, L188C, S190I, and D486S;

(9) combination of S155C, S190I, S290C, and D486S;

(10) combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S,E487Q, and D489S; and

(11) combination of T54H, S155C, S190I, S290C, and V296I.

In some specific embodiments, the present disclosure provides a RSV Fmutant comprising a combination of introduced mutations, wherein themutant comprises a combination of mutations in any of the mutantsprovided in Tables 4, 5, and 6. RSV F mutants provided in Tables 4, 5,and 6 are based on the same native F0 sequence of RSV A2 strain withthree naturally occurring substitutions at positions 102, 379, and 447(SEQ ID NO:3). The same introduced mutations in each of the mutants canbe made to a native F0 polypeptide sequence of any other RSV subtype orstrain to arrive at different RSV F mutants, such as a native F0polypeptide sequence set forth in any of the SEQ ID NOs:1, 2, 4, 6, and81-270. RSV F mutants that are based on a native F0 polypeptide sequenceof any other RSV subtype or strain and comprise any of the combinationof mutations are also within the scope of the invention.

In some other particular embodiments, the present invention provides aRSV F mutant, wherein the mutant comprises a cysteine (C) at position103 (103C) and at position 148 (148C), an isoleucine (1) at position 190(190I), and a serine (S) at position 486 (486S), and wherein the mutantcomprises a F1 polypeptide and a F2 polypeptide selected from the groupconsisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:41and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:42;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:41and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:42;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 43and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:44;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:43and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:44;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 45and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:46;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:45and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:46;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 47and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:48;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:47and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:48;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 49and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:50;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:49and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:50.

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:279 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:280;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:279and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:280;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:281 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:282;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:281and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:282;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:283 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:284;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:283and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:284;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:285 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:286;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:285and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:286;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:287 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:288;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:287and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:288;

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:289 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:290; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:289and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:290.

In some other particular embodiments, the present invention provides aRSV F mutant, wherein the mutant comprises a histidine (H) at position54, a cysteine (C) at positions 103 and 148, a isoleucine (I) atpositions 190 and 296, and a serine (S) at position 486, and wherein themutant comprises a F1 polypeptide and a F2 polypeptide selected from thegroup consisting of:

(1) F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 51and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:52;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:51and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:52;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:53and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:54;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:53and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:54;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:55and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:56;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:55and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:57and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:57and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:58;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:59and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:60;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:291 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:292;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:291and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:292;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:293 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:294;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:293and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:294;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:295 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:296;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:295and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:296;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:297 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:298;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:297and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:298;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:299 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:300;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:299and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:300;

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:301 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:302; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:301and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:302.

In some other particular embodiments, the present invention provides aRSV F mutant, wherein the mutant comprises a histidine (H) at position54, a cysteine (C) at positions 55 and 188, and a serine (S) at position486, and wherein the mutant comprises a F1 polypeptide and a F2polypeptide selected from the group consisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:61and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:62;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:61 anda F1 polypeptide comprising an amino acid sequence that is at least 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO:62;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:63and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:64;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:63and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:64;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:65and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:66;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:65and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:66;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:67and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:68;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:67and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:68;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:69and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:70;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:69and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:70;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:303 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:304;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:303and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:304;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:305 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:306;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:305and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:306;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:307 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:308;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:307and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:308;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:309 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:310;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:309and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:310;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:311 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:312;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:311and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:312.

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:313 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:314; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:313and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:314.

In some other particular embodiments, the present invention provides aRSV F mutant, wherein the mutant comprises a histidine (H) at position54, a cysteine (C) at positions 55 and 188, an isoleucine (1) atposition 190 (190I), and a serine (S) at position 486, and wherein themutant comprises a F1 polypeptide and a F2 polypeptide selected from thegroup consisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:71and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:72;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:71and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:72;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:73and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:74;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:73and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:74;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:75and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:76;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:75and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:76;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:77and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:78;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:77and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:78;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:79and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:80;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:79and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:80;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:315 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:316;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:315and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:316;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:317 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:318;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:317and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:318;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:319 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:320;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:319and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:320;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:321 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:322;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:321and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:322;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:323 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:324;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:323and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:324.

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:325 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:326; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:325and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:326.

The amino acid sequence of the F2 polypeptide and F1 polypeptide ofexemplary RSV F mutants provided by the present disclosure is providedin Tables C-F.

In several embodiments, a foldon domain is linked to a RSV F mutantdescribed herein above, wherein the foldon domain is linked to theC-terminus of the F1 polypeptide and comprises the amino acid sequenceof SEQ ID NO:40.

The RSV F protein mutants provided by the present disclosure can beprepared by routine methods known in the art, such as by expression in arecombinant host system using a suitable vector. Suitable recombinanthost cells include, for example, insect cells, mammalian cells, aviancells, bacteria, and yeast cells. Examples of suitable insect cellsinclude, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2cells, and High Five cells (a clonal isolate derived from the parentalTrichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples ofsuitable mammalian cells include Chinese hamster ovary (CHO) cells,human embryonic kidney cells (HEK293 or Expi 293 cells, typicallytransformed by sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-Tcells, Vero cells, and HeLa cells. Suitable avian cells include, forexample, chicken embryonic stem cells (e.g., EBx® cells), chickenembryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts(e.g. ELL-O), and duck cells. Suitable insect cell expression systems,such as baculovirus-vectored systems, are known to those of skill in theart and described in, e.g., Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987). Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. Avian cellexpression systems are also known to those of skill in the art anddescribed in, e.g., U.S. Pat. Nos. 5,340,740; 5,656,479; 5,830,510;6,114,168; and 6,500,668. Similarly, bacterial and mammalian cellexpression systems are also known in the art and described in, e.g.,Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths,London.

A number of suitable vectors for expression of recombinant proteins ininsect or mammalian cells are well-known and conventional in the art.Suitable vectors can contain a number of components, including, but notlimited to one or more of the following: an origin of replication; aselectable marker gene; one or more expression control elements, such asa transcriptional control element (e.g., a promoter, an enhancer, aterminator), and/or one or more translation signals; and a signalsequence or leader sequence for targeting to the secretory pathway in aselected host cell (e.g., of mammalian origin or from a heterologousmammalian or non-mammalian species). For example, for expression ininsect cells a suitable baculovirus expression vector, such as pFastBac(Invitrogen), is used to produce recombinant baculovirus particles. Thebaculovirus particles are amplified and used to infect insect cells toexpress recombinant protein. For expression in mammalian cells, a vectorthat will drive expression of the construct in the desired mammalianhost cell (e.g., Chinese hamster ovary cells) is used.

The RSV F protein mutant polypeptides can be purified using any suitablemethods. For example, methods for purifying RSV F protein mutantpolypeptides by immunoaffinity chromatography are known in the art.Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitablemethods for purifying desired proteins including precipitation andvarious types of chromatography, such as hydrophobic interaction, ionexchange, affinity, chelating and size exclusion are well-known in theart. Suitable purification schemes can be created using two or more ofthese or other suitable methods. If desired, the RSV F protein mutantpolypeptides can include a “tag” that facilitates purification, such asan epitope tag or a histidine (HIS) tag. Such tagged polypeptides canconveniently be purified, for example from conditioned media, bychelating chromatography or affinity chromatography.

C. NUCLEIC ACIDS ENCODING RSV F PROTEIN MUTANTS

In another aspect, the present invention provides nucleic acid moleculesthat encode a RSV F protein mutant described herein above. These nucleicacid molecules include DNA, cDNA, and RNA sequences. Nucleic acidmolecules that encode only a F2 polypeptide or only a F1 polypeptide ofa RSV F mutant are also encompassed by the invention. The nucleic acidmolecule can be incorporated into a vector, such as an expressionvector.

In some embodiments, the nucleic acid molecule encodes a precursor F0polypeptide that, when expressed in an appropriate cell, is processedinto a disclosed RSV F mutant. In some embodiments, the nucleic acidmolecule encodes a precursor F0 polypeptide that, when expressed in anappropriate cell, is processed into a disclosed RSV F mutant, whereinthe precursor F0 polypeptide includes, from N- to C-terminus, a signalpeptide, a F2 polypeptide, a Pep27 polypeptide, and a F1 polypeptide. Insome embodiments, the Pep27 polypeptide comprises the amino acidsequence set forth at positions 110-136 of any of the amino acidsequences of SEQ ID NOs:1, 2, 4, 6, and 81-270, wherein the amino acidpositions correspond to the amino acid sequence of SEQ ID NO:1. In someembodiments, the signal peptide comprises the amino acid sequence setforth at positions 1-25 of any one of the amino acid sequences of SEQ IDNOs: 1, 2, 4, 6, and 81-270, wherein the amino acid positions correspondto the amino acid sequence of a reference of SEQ ID NO:1.

In some embodiments, the nucleic acid molecule encodes a mutant selectedfrom the group consisting of:

(1) a mutant comprising at least one engineered disulfide mutation;

(2) a mutant comprising at least one cavity filing mutation;

(3) a mutant comprising at least one electrostatic mutation;

(4) a mutant comprising at least one engineered disulfide mutation andat least one cavity filing mutation;

(5) a mutant comprising at least one engineered disulfide mutation andat least one electrostatic mutation;

(6) a mutant comprising at least one cavity filing mutation and at leastone electrostatic mutation; and

(7) a mutant comprising at least one engineered disulfide mutation andat least one electrostatic mutation, at least one cavity filingmutation, and at least one electrostatic mutation.

In some specific embodiments, the present disclosure provides a nucleicacid molecule which encodes a mutant selected from the group consistingof:

(1) a mutant comprising a combination of substitutions 103C, 148C, 190I,and 486S;

(2) a mutant comprising a combination of substitutions 54H, 55C, 188C,and 486S;

(3) a mutant comprising a combination of substitutions 54H, 103C, 148C,190I, 296I, and 486S;

(4) a mutant comprising a combination of substitutions 54H, 55C, 142C,188C, 296I, and 371C;

(5) a mutant comprising a combination of amino acid substitutions 55C,188C, and 486S;

(6) a mutant comprising a combination of amino acid substitutions 54H,55C, 188C, and 190I;

(7) a mutant comprising a combination of amino acid substitutions 55C,188C, 190I, and 486S;

(8) a mutant comprising a combination of amino acid substitutions 54H,55C, 188C, 190I, and 486S;

(9) a mutant comprising a combination of amino acid substitutions 155C,190I, 290C, and 486S;

(10) a mutant comprising a combination of amino acid substitutions 54H,55C, 142C, 188C, 296I, 371C, 486S, 487Q, and 489S; and

(11) a mutant comprising a combination of amino acid substitutions 54H,155C, 190I, 290C, and 296I.

In some particular embodiments, the nucleic acid molecule encodes a RSVF mutant, wherein the mutant comprises a cysteine (C) at position 103(103C) and at position 148 (148C), an isoleucine (1) at position 190(190I), and a serine (S) at position 486 (486S), and wherein the mutantcomprises a F1 polypeptide and a F2 polypeptide selected from the groupconsisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:41and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:42;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:41and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:42;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 43and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:44;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:43and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:44;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 45and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:46;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:45and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:46;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 47and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:48;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:47and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:48;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 49and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:50;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:49and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:50.

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:279 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:280;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:279and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:280;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:281 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:282;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:281and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:282;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:283 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:284;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:283and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:284;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:285 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:286;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:285and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:286;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:287 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:288;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:287and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:288;

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:289 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:290; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:289and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:290.

In some other particular embodiments, the nucleic acid molecule encodesa RSV F mutant, wherein the mutant comprises a histidine (H) at position54, a cysteine (C) at positions 103 and 148, a isoleucine (I) atpositions 190 and 296, and a serine (S) at position 486, and wherein themutant comprises a F1 polypeptide and a F2 polypeptide selected from thegroup consisting of:

(1) F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 51and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:52;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:51and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:52;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:53and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:54;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:53and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:54;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:55and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:56;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:55and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:57and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:57and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:58;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:59and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:60;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:291 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:292;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:291and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:292;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:293 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:294;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:293and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:294;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:295 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:296;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:295and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:296;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:297 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:298;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:297and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:298;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:299 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:300;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:299and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:300;

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:301 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:302; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:301and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:302.

In some other particular embodiments, the nucleic acid molecule encodesa RSV

F mutant, wherein the mutant comprises a histidine (H) at position 54, acysteine (C) at positions 55 and 188, and a serine (S) at position 486,and wherein the mutant comprises a F1 polypeptide and a F2 polypeptideselected from the group consisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:61and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:62;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:61and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:62;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:63and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:64;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:63and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:64;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:65and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:66;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:65and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:66;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:67and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:68;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:67and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:68;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:69and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:70;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:69and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:70;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:303 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:304;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:303and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:304;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:305 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:306;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:305and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:306;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:307 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:308;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:307and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:308;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:309 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:310;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:309and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:310;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:311 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:312;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:311and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:312.

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:313 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:314; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:313and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:314.

In some other particular embodiments, the nucleic acid molecule encodesa RSV F mutant, wherein the mutant comprises a histidine (H) at position54, a cysteine (C) at positions 55 and 188, an isoleucine (1) atposition 190 (190I), and a serine (S) at position 486, and wherein themutant comprises a F1 polypeptide and a F2 polypeptide selected from thegroup consisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:71and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:72;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:71and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:72;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:73and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:74;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:73and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:74;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:75and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:76;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:75and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:76;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:77and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:78;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:77and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:78;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:79and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:80;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:79and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:80;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:315 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:316;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:315and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:316;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:317 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:318;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:317and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:318;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:319 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:320;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:319and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:320;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:321 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:322;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:321and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:322;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:323 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:324;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:323and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:324.

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:325 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:326; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:325and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:326.

In some specific embodiments, the present disclosure provides a nucleicacid molecule, which encodes a mutant selected from the group consistingof:

(1) a mutant comprising amino acids 26-513 of SEQ ID NO:19;

(2) a mutant comprising amino acids 26-513 of SEQ ID NO:20; and

(3) a mutant comprising amino acids 26-513 of SEQ ID NO:21.

In some other specific embodiments, the present disclosure provides anucleic acid molecule encoding a RSV F protein mutant, or a degeneratevariant thereof, wherein the nucleic acid molecule comprises anucleotide sequence selected from the group consisting of:

(1) a nucleotide sequence comprising nucleotides 76-1539 of SEQ ID NO:8;

(2) a nucleotide sequence comprising nucleotides 76-1539 of SEQ ID NO:9;

(3) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:10;

(4) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:11;

(5) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:12;

(6) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:13;

(7) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:14;

(8) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:15;

(9) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:16;

(10) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:17; and

(11) a nucleotide sequence comprising nucleotides 76-1539 of SEQ IDNO:18.

D. COMPOSITIONS COMPRISING A RSV F PROTEIN MUTANT; COMPOSITIONSCOMPRISING A NUCLEIC ACID ENCODING A RSV F PROTEIN MUTANT

In another aspect, the invention provides compositions that comprise (1)a RSV F protein mutant described in the disclosure, or (2) a nucleicacid molecule or vector encoding such a RSV F protein mutant.

In some embodiments, the composition is an immunogenic compositioncapable of eliciting an immune response against the F protein of RSV ina subject. In some particular embodiments, the immunogenic compositionis a pharmaceutical composition, which comprises a RSV F protein mutantprovided by the present disclosure and a pharmaceutically acceptablecarrier.

In still other embodiments, the pharmaceutical composition is a vaccine.The immunogenic component in the vaccine may be (1) a RSV F proteinmutant described herein, (2) a nucleic acid encoding such as a RSV Fprotein mutant, or (3) a vector for expressing such a RSV F proteinmutant.

In some particular embodiments, the vaccine comprises a RSV F proteinmutant, wherein the mutant comprises a cysteine (C) at position 103(103C) and at position 148 (148C), an isoleucine (1) at position 190(190I), and a serine (S) at position 486 (486S), and wherein the mutantcomprises a F1 polypeptide and a F2 polypeptide selected from the groupconsisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:41and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:42;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:41and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:42;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 43and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:44;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:43and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:44;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 45and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:46;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:45and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:46;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 47and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:48;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:47and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:48;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 49and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:50;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:49and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:50.

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:279 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:280;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:279and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:280;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:281 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:282;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:281and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:282;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:283 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:284;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:283and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:284;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:285 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:286;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:285and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:286;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:287 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:288;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:287and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:288;

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:289 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:290; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:289and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:290.

In some other particular embodiments, the vaccine comprises a RSV Fmutant, wherein the mutant comprises a histidine (H) at position 54, acysteine (C) at positions 103 and 148, a isoleucine (I) at positions 190and 296, and a serine (S) at position 486, and wherein the mutantcomprises a F1 polypeptide and a F2 polypeptide selected from the groupconsisting of:

(1) F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 51and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:52;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:51and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:52;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:53and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:54;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:53and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:54;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:55and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:56;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:55and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:57and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:58;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:57and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:58;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:59and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:60;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:60;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:291 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:292;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:291and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:292;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:293 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:294;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:293and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:294;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:295 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:296;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:295and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:296;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:297 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:298;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:297and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:298;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:299 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:300;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:299and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:300;

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:301 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:302; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:301and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:302.

In some other particular embodiments, the vaccine comprises a RSV Fmutant, wherein the mutant comprises a histidine (H) at position 54, acysteine (C) at positions 55 and 188, and a serine (S) at position 486,and wherein the mutant comprises a F1 polypeptide and a F2 polypeptideselected from the group consisting of: (1) a F2 polypeptide comprisingthe amino acid sequence of SEQ ID NO:61 and a F1 polypeptide comprisingthe amino acid sequence of SEQ ID NO:62;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:61and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:62;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:63and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:64;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:63and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:64;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:65and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:66;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:65and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:66;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:67and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:68;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:67and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:68;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:69and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:70;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:69and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:70;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:303 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:304;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:303and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:304;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:305 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:306;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:305and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:306;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:307 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:308;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:307and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:308;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:309 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:310;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:309and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:310;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:311 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:312;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:311and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:312.

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:313 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:314; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:313and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:314.

In some other particular embodiments, the vaccine comprises a RSV Fmutant, wherein the mutant comprises a histidine (H) at position 54, acysteine (C) at positions 55 and 188, an isoleucine (1) at position 190(190I), and a serine (S) at position 486, and wherein the mutantcomprises a F1 polypeptide and a F2 polypeptide selected from the groupconsisting of:

(1) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:71and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:72;

(2) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:71and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:72;

(3) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:73and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:74;

(4) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:73and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:74;

(5) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:75and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:76;

(6) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:75and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:76;

(7) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:77and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:78;

(8) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:77and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:78;

(9) a F2 polypeptide comprising the amino acid sequence of SEQ ID NO:79and a F1 polypeptide comprising the amino acid sequence of SEQ ID NO:80;

(10) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:79 anda F1 polypeptide comprising an amino acid sequence that is at least 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO:80;

(11) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:315 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:316;

(12) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:315and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:316;

(13) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:317 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:318;

(14) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:317and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:318;

(15) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:319 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:320;

(16) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:319and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:320;

(17) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:321 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:322;

(18) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:321and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:322;

(19) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:323 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:324;

(20) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:323and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:324.

(21) a F2 polypeptide comprising the amino acid sequence of SEQ IDNO:325 and a F1 polypeptide comprising the amino acid sequence of SEQ IDNO:326; and

(22) a F2 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:325and a F1 polypeptide comprising an amino acid sequence that is at least97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:326.

In some embodiments, a composition, such as a pharmaceutical compositionor a vaccine, comprises two or more different RSV F mutants. The two ormore different RSV F mutants may comprise the same introduced amino acidmutations but comprise a F1 polypeptide and F2 polypeptide fromdifferent RSV strains or subtypes. The two or more different RSV Fmutants may comprise different introduced amino acid mutations.

In some embodiments, the composition comprises two different mutantscomprising the same introduced amino acid mutations, wherein one of themutant comprises a F1 polypeptide and F2 polypeptide from RSV subtype Aand wherein the other mutant comprises a F1 polypeptide and F2polypeptide from RSV subtype B. In some specific embodiments, the twodifferent mutants comprise the same combination of amino acidsubstitutions selected from the group consisting of:

(1) a combination of amino acid substitutions 103C, 148C, 190I, and486S;

(2) a combination of amino acid substitutions 54H, 55C, 188C, and 486S;

(3) a combination of amino acid substitutions 54H, 103C, 148C, 190I,296I, and 486S;

(4) a combination of amino acid substitutions 54H, 55C, 142C, 188C,296I, and 371C;

(5) a combination of amino acid substitutions 55C, 188C, and 486S;

(6) a combination of amino acid substitutions 54H, 55C, 188C, and 190I;

(7) a combination of amino acid substitutions 55C, 188C, 190I, and 486S;

(8) a combination of amino acid substitutions 54H, 55C, 188C, 190I, and486S;

(9) a combination of amino acid substitutions 155C, 190I, 290C, and486S;

(10) a combination of amino acid substitutions 54H, 55C, 142C, 188C,296I, 371C, 486S, 487Q, and 489S; and

(11) a combination of amino acid substitutions 54H, 155C, 190I, 290C,and 296I.

In addition to the immunogenic component, the vaccine may furthercomprise an immunomodulatory agent, such as an adjuvant. Examples ofsuitable adjuvants include aluminum salts such as aluminum hydroxideand/or aluminum phosphate; oil-emulsion compositions (or oil-in-watercompositions), including squalene-water emulsions, such as MF59 (seee.g., WO 90/14837); saponin formulations, such as, for example, QS21 andImmunostimulating Complexes (ISCOMS) (see e.g., U.S. Pat. No. 5,057,540;WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial ormicrobial derivatives, examples of which are monophosphoryl lipid A(MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containingoligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof,such as E. coli heat labile enterotoxin LT, cholera toxin CT, and thelike. It is also possible to use vector-encoded adjuvant, e.g., by usingheterologous nucleic acid that encodes a fusion of the oligomerizationdomain of C4-binding protein (C4 bp) to the antigen of interest (e.g.,Solabomi et al., 2008, Infect Immun 76: 3817-23). In certain embodimentsthe compositions hereof comprise aluminum as an adjuvant, e.g., in theform of aluminum hydroxide, aluminum phosphate, aluminum potassiumphosphate, or combinations thereof, in concentrations of 0.05-5 mg,e.g., from 0.075-1.0 mg, of aluminum content per dose.

E. USES OF THE RSV F PROTEIN MUTANTS, NUCLEIC ACID MOLECULES, ANDCOMPOSITIONS

The present disclosure also relates to use of a RSV F protein mutant,nucleic acids encoding a RSV F protein mutant, or vectors for expressinga RSV F protein mutant, or compositions comprising a RSV F proteinmutant or nucleic acids.

In one aspect, the disclosure provides use of a RSV F protein mutant,nucleic acids encoding a RSV F protein mutant, or vectors for expressinga RSV F protein mutant, or compositions comprising a RSV F proteinmutant or nucleic acids as a medicament, or in the manufacture of amedicament, for eliciting an immune response against RSV or forpreventing or elevating RSV infection in a subject.

In other aspects, the present disclosure provides a method of elicitingan immune response against RSV in a subject, such as a human, comprisingadministering to the subject an effective amount of a RSV F proteinmutant, a nucleic acid molecule encoding a RSV F protein mutant, or acomposition comprising a RSV F protein mutant or nucleic acid molecule.The present disclosure also provides a method of preventing RSVinfection in a subject, comprising administering to the subject aneffective amount of a pharmaceutical composition, such as a vaccine,comprising a RSV F protein mutant, a nucleic acid encoding a RSV Fprotein mutant, or a vector expressing a RSV F protein mutant. In someparticular embodiments, the pharmaceutical composition comprises a RSV Fprotein mutant. In some embodiments of the methods provided hereinabove, the subject is a human. In some particular embodiments, the humanis a child, such as an infant. In some other particular embodiments, thehuman is a woman, particularly a pregnant woman.

The composition may be administered to the subject with or withoutadministration of an adjuvant. The effective amount administered to thesubject is an amount that is sufficient to elicit an immune responseagainst an RSV antigen, such as RSV F protein, in the subject. Subjectsthat can be selected for treatment include those that are at risk fordeveloping an RSV infection because of exposure or the possibility ofexposure to RSV. Because nearly all humans are infected with RSV by theage of 2, the entire birth cohort is included as a relevant populationfor immunization. This could be done, for example, by beginning animmunization regimen anytime from birth to 6 months of age, from 6months of age to 5 years of age, in pregnant women (or women ofchild-bearing age) to protect their infants by passive transfer ofantibody, family members of newborn infants or those still in utero, andsubjects greater than 50 years of age. Subjects at greatest risk of RSVinfection with severe symptoms (e.g. requiring hospitalization) includechildren with prematurity, bronchopulmonary dysplasia, and congenitalheart disease.

Administration of the compositions provided by the present disclosure,such as pharmaceutical compositions, can be carried out using standardroutes of administration. Non-limiting embodiments include parenteraladministration, such as intradermal, intramuscular, subcutaneous,transcutaneous, mucosal, or oral administration.

The total dose of the composition provided to a subject during oneadministration can be varied as is known to the skilled practitioner.

It is also possible to provide one or more booster administrations ofone or more of the vaccine compositions. If a boosting vaccination isperformed, typically, such a boosting vaccination will be administeredto the same subject at a moment between one week and 10 years,preferably between two weeks and six months, after administering thecomposition to the subject for the first time (which is in such casesreferred to as “priming vaccination”). In alternative boosting regimens,it is also possible to administer different vectors, e.g., one or moreadenovirus, or other vectors such as modified vaccinia virus of Ankara(MVA), or DNA, or protein, to the subject after the priming vaccination.It is, for instance, possible to administer to the subject a recombinantviral vector hereof as a prime, and boosting with a compositioncomprising RSV F protein.

In certain embodiments, the administration comprises a primingadministration and at least one booster administration. In certain otherembodiments, the administration is provided annually. In still otherembodiments, the administration is provided annually together with aninfluenza vaccine.

The vaccines provided by the present disclosure may be used togetherwith one or more other vaccines. For example, in adults they may be usedtogether with an influenza vaccine, Prevnar, tetanus vaccine, diphtheriavaccine, and pertussis vaccine. For pediatric use, vaccines provided bythe present disclosure may be used with any other vaccine indicated forpediatric patients.

TABLE A Non-consensus amino acid residues among F protein sequences fromselected RSV A strains. Strain Name (GenBank) RSVA/Homo RSVA/Homo Aminosapiens/USA/L A/WI/629- sapiens/USA/90I- Acid A2 A2_21/2013 4071/98TX-79223 BE08-5146 Tracy RSV-4 06-000827 226A-01/1990 Position (138251)(AHX57185) (AEQ63520) (AGG39418) (AFM55563) (AGG39397) (AEO45850)(AFM55442) (AHY21463) 4 L P P P P P P P P 6 L L L L L I L L L 8 A T T TT A T T T 15 L L L L L L L F F 16 T A A A A I T A A 20 F L L L L F F L L25 G S S S S S S S S 59 I I I I I I I I V 101 P P P P Q T P P P 102 P AA A A A A A A 103 T A A A A A A A A 105 N S N N S N N N N 122 A T T T TA T T T 124 K N N T N K N N N 125 T T T T T T N N T 129 L L V L L L L LL 152 V I I I I I I I I 276 N S N N N N N N N 356 E E E E E D E E E 379I V V V V V V V V 384 V I I T I I V I I 447 M V V V V V V V V 518 A A AA A A V V A 540 S A S S S S L L S 547 L L L L L L L F L 562 D D D D D DD E D 574 N N N S N N N N N

TABLE B Non-consensus amino acid residues among F protein sequences fromselected RSV B strains. Strain Name (GenBank) RSVB/Homo Aminosapiens/PER/ Acid 18537 FPP00592/2011 NH1144 TX-79247 CH-18537 NH1125TX-79222 TX-60567 Position (138250) (AHV80758) (AFD34260) (AGG39514)(AGG39487) (AFI25251) (AGG39523) (AGG39502) 5 I I I I I I I V 9 S S S SS S I S 17 V I I I V I I I 45 F F F L F F F F 65 K K K K K K T K 102 A AA V A A A A 123 K K K K K K K N 152 I I I I M I I I 185 V V V V I V V V202 R Q Q Q R Q Q Q 209 Q Q K Q Q Q Q Q 226 M K K K K K K K 234 T T T TT T T A 292 I I I I I M I I 326 I I I T I I I I 371 N N N Y N N N N 402I I V I I I I I 518 T T T T T T V T 529 T A A A T V A A

TABLE C Variants of Mutant pXCS847 Comprising IntroducedMutations T103C, I148C, S190I, and D486S F2 PolypeptideAmino Acid Sequence F1 Polypeptide Mutant ID SEQ ID (residues 26-109)SEQ ID Amino Acid Sequence (residues 137-513) pXCS847  41QNITEEFYQSTCSAVSKGY  42 FLGFLLGVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKALSALRTGWYTSVITIELSN VVSLSNGVSVLT

KVLDLKNYIDKQLLPIVNKQSCSISNIE IKENKCNGTDAKVKLIKQETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLS LDKYKNAVTELQLLMQSTPLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPS

EFDASISQVNEKINQSLAF IRKSDELL 847-138251  43 QNITEEFYQSTCSAVSKGY  44FLGFLLGVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKA (A2) LSALRTGWYTSVITIELSNVVSLSNGVSVLT

KVLDLKNYIDKQLLPIVNKQSCSISNIE IKENKCNGTDAKVKLIKQETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLS LDKYKNAVTELQLLMQSTPLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV PCNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPS

EFDASISQVNEKINQSLAF IRKSDELL 847-57185 (A)  45 QNITEEFYQSTCSAVSKGY  46FLGFLLGVGSA

ASGIAVSKVLHLEGEVNKIKSALLSTNKA (Ontario) LSALRTGWYTSVITIELSN VVSLSNGVSVLT

KVLDLKNYIDKQLLPIVNKQSCSISNIE IKENKCNGTDAKVKLIKQETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLS LDKYKNAVTELQLLMQSTPLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV ACNSRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPS

EFDASISQVNEKINQSLAF IRKSDELL 847-138250(B)  47 QNITEEFYQSTCSAVSRGY  48FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKA FSALRTGWYTSVITIELSNVVSLSNGVSVLT

KVLDLKNYINNRLLPIVNQQSCRISNIE IKETKCNGTDTKVKLIKQETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLS LDKYKNAVTELQLLMQNTPLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEG KNLYVKGEPIINYYDPLVFPS

EFDASISQVNEKINQSLAF IRRSDELL 847-80758 (B)  49 QNITEEFYQSTCSAVSRGY  50FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKA (Buenos Aires)FSALRTGWYTSVITIELSN VVSLSNGVSVLT

KVLDLKNYINNQLLPIVNQQSCRISNI IKETKCNGTTKVKLIKQEDETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELL LDKYKNAVTELQLLMQNTPSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAY ACNNRARRVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLE KNLYVKGEPIINYYDPLVFPS

EFDASISQVNEKINQSL AFIRRSDELL 847-AFM55442 279 QNITEEFYQSTCSAVSKGY 280FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKA (A) LSALRTGWYTSVITIELSNVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIE IKENKCNGTDAKVKLIKQETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLS LDKYKNAVTELQLLMQSTPLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL 847-AFM95376 281QNITEEFYQSTCSAVSKGY 282 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKA (A)LSALRTGWYTSVITIELSN VVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL 847-AEQ63520 283QNITEEFYQSTCSAVSKGY 284 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKA (A)LSALRTGWYTSVITIELSN VVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL 847-AFD34260 285QNITEEFYQSTCSAVSRGY 286 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKA (B)FSALRTGWYTSVITIELSN VVSLSNGVSVLTIKVLDLKNYINNQLLPIVNKQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL 847-BAE96918 287QNITEEFYQSTCSAVSRGY 288 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKA (B)FSALRTGWYTSVITIELSN VVSLSNGVSVLTIKVLDLKNYINNQLLPIVNQQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIMKEEVLAYV ACNNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL 847-AFD34265 289QNITEEFYQSTCSAVSRGY 290 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKA (B)LSALRTGWYTSVITIELSN VVSLSNGVSVLTIKVLDLKNYINNQLLPIVNQQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQKNSRLLEIAREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV ACNNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL

TABLE D Variant of Mutant pXCS851 Comprising IntroducedMutations T54H, T103C, I148C, S190I, V296I, D486S F2 PolypeptideAmino Acid Sequence F1 Polypeptide Mutant ID SEQ ID (residued 26-109)SEQ ID Amino Acid Sequence (residues 137-513) pXCS851  51QNITEEFYQSTCSAVSKGY  52 FLGFLLGVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNLSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLIKENKCNGTDAKVKLIKQE LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNLDKYKNAVTELQLLMQSTP VQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTACNNRARR NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDEL L GI-138251 (A2)  53QNITEEFYQSTCSAVSKGY  54 FLGFLLGVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNLSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLIKENKCNGTDAKVKLIKQE LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNLDKYKNAVTELQLLMQSTP VQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTPCNNRARR NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDEL L GI-57185 (A)  55QNITEEFYQSTCSAVSKGY  56 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSN(Ontario) LSALRTGWYHSVITIELSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRL IKENKCNGTDAKVKLIKQELEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSN LDKYKNAVTELQLLMQSTPVQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTT ACNSRARRNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDEL L GI-138250 (B)  57QNITEEFYQSTCSAVSRGY  58 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNFSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLIKETKCNGTDTKVKLIKQE LEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNLDKYKNAVTELQLLMQNTP VQIVRQQSYSIMSIIKEEILAYVVQLPIYGVIDTPCWKLHTSPLCTTACNNRARR NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDEL L GI-80758 (B)  59QNITEEFYQSTCSAVSRGY  60 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSN(Buenos Aires) FSALRTGWYHSVITIELSNGVSVLTIKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRL IKETKCNGTDTKVKLIKQELEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSN LDKYKNAVTELQLLMQNTPVQIVRQQSYSIMSIIKEEILAYVVQLPIYGVIDTPCWKLHTSPLCTT ACNNRARRNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDEL L 851-AFM55442 291QNITEEFYQSTCSAVSKGY 292 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSN(A) LSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLIKENKCNGTDAKVKLIKQE LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNLDKYKNAVTELQLLMQSTP VQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTACNNRARR NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDEL L 851-AFM95376 293QNITEEFYQSTCSAVSKGY 294 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSN(A) LSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLIKENKCNGTDAKVKLIKQE LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNLDKYKNAVTELQLLMQSTP VQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTACNNRARR NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDEL L 851-AEQ63520 295QNITEEFYQSTCSAVSKGY 296 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSN(A) LSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLIKENKCNGTDAKVKLIKQE LEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNLDKYKNAVTELQLLMQSTP VQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTACNNRARR NTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDEL L 851-AFD34260 297QNITEEFYQSTCSAVSRGY 298 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSN(B) FSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYINNQLLPIVNKQSCRISNIETVIEFQQKNSRLIKETKCNGTDTKVKLIKQE LEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNLDKYKNAVTELQLLMQNTP VQIVRQQSYSIMSIIKEEILAYVVQLPIYGVIDTPCWKLHTSPLCTTACNNRARR NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDEL L 851-BAE96918 299QNITEEFYQSTCSAVSRGY 300 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSN(B) FSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLIKETKCNGTDTKVKLIKQE LEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNLDKYKNAVTELQLLMQNTP VQIVRQQSYSIMSIMKEEILAYVVQLPIYGVIDTPCWKLHTSPLCTTACNNRARR NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDEL L 851-AFD34265 301QNITEEFYQSTCSAVSRGY 302 FLGFLLGVGSACASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSN(B) LSALRTGWYHSVITIELSN GVSVLTIKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLIKETKCNGTDTKVKLIKQE LEIAREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNLDKYKNAVTELQLLMQNTP VQIVRQQSYSIMSIIKEEILAYVVQLPIYGVIDTPCWKLHTSPLCTTACNNRARR NIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDEL L

TABLE E Variant of Mutants pXCS852 Comprising IntroducedMutations T54H, S55C, L188C, D486S F2 Polypeptide Amino Acid SequenceF1 Polypeptide Mutant ID SEQ ID (residues 26-109) SEQ IDAmino Acid Sequence (residues 137-513) pXCS852  61 QNITEEFYQSTCSAVSKGY 62 FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA LSALRTGWYHCVITIELSNVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIE IKENKCNGTDAKVKLIKQETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLS LDKYKNAVTELQLLMQSTPLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV ATNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL GI-138251 (A2)  63QNITEEFYQSTCSAVSKGY  64 FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKALSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV PTNNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL GI-57185 (A)  65QNITEEFYQSTCSAVSKGY  66 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKA(Ontario) LSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV AANSRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL GI-138250 (B)  67QNITEEFYQSTCSAVSRGY  68 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAFSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYINNRLLPIVNQQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL GI-80758 (B)  69QNITEEFYQSTCSAVSRGY  70 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKA(Buenos Aires) FSALRTGWYHCVITIELSNVVSLSNGVSVCTSKVLDLKNYINNQLLPIVNQQSCRISNIE IKETKCNGTDTKVKLIKQETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLS LDKYKNAVTELQLLMQNTPLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL 852-AFM55442 303QNITEEFYQSTCSAVSKGY 304 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKA (A)LSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL 852-AFM95376 305QNITEEFYQSTCSAVSKGY 306 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKA (A)LSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL 852-AEQ63520 307QNITEEFYQSTCSAVSKGY 308 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKA (A)LSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIEIKENKCNGTDAKVKLIKQE TVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLDKYKNAVTELQLLMQSTP LINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAF IRKSDELL 852-AFD34260 309QNITEEFYQSTCSAVSRGY 310 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKA (B)FSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYINNQLLPIVNKQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL 852-BAE96918 311QNITEEFYQSTCSAVSRGY 312 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKA (B)FSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYINNQLLPIVNQQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIMKEEVLAYV AANNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL 852-AFD34265 313QNITEEFYQSTCSAVSRGY 314 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKA (B)LSALRTGWYHCVITIELSN VVSLSNGVSVCTSKVLDLKNYINNQLLPIVNQQSCRISNIEIKETKCNGTDTKVKLIKQE TVIEFQQKNSRLLEIAREFSVNAGVTTPLSTYMLTNSELLSLDKYKNAVTELQLLMQNTP LINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYV AANNRARRVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAF IRRSDELL

TABLE F Variant of Mutant pXCS855 Comprising IntroducedMutations T54H, S55C, L188C, S190I, D486S F2 PolypeptideAmino Acid Sequence F1 Polypeptide Mutant ID SEQ ID (residues 26-109)SEQ ID Amino Acid Sequence (residues 137-513) pXCS855  71QNITEEFYQSTCSAVSKGY  72 FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVLSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIKENKCNGTDAKVKLIKQE IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINLDKYKNAVTELQLLMQSTP DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLP ATNNRARRLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELL 855-GI138251  73QNITEEFYQSTCSAVSKGY  74 FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAV (A2)LSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIKENKCNGTDAKVKLIKQE IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINLDKYKNAVTELQLLMQSTP DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLP PTNNRARRLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELL 855-GI57185  75QNITEEFYQSTCSAVSKGY  76 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAV(A) (Ontario) LSALRTGWYHCVITIELSNVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETV IKENKCNGTDAKVKLIKQEIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLIN LDKYKNAVTELQLLMQSTPDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLP AANSRARRLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELL 855-0138250  77QNITEEFYQSTCSAVSRGY  78 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAV (B)FSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYINNRLLPIVNQQSCRISNIETVIKETKCNGTDTKVKLIKQE IEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINLDKYKNAVTELQLLMQN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDELL 855-GI80758  79QNITEEFYQSTCSAVSRGY  80 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAV (B)FSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYINNQLLPIVNQQSCRISNIETV(Buenos Aires) IKETKCNGTDTKVKLIKQEIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN LDKYKNAVTELQLLMQNDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDELL 855-AFM55442 315QNITEEFYQSTCSAVSKGY 316 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAV (A)LSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIKENKCNGTDAKVKLIKQ IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINELDKYKNAVTELQLLMQS DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELL 855-AFM95376 317QNITEEFYQSTCSAVSKGY 318 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAV (A)LSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIKENKCNGTDAKVKLIKQ IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINELDKYKNAVTELQLLMQS DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELL 855-AEQ63520 319QNITEEFYQSTCSAVSKGY 320 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAV (A)LSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIKENKCNGTDAKVKLIKQ IEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINELDKYKNAVTELQLLMQS DMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELL 855-AFD34260 321QNITEEFYQSTCSAVSRGY 322 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAV (B)FSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYINNQLLPIVNKQSCRISNIETVIKETKCNGTDTKVKLIKQE IEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINLDKYKNAVTELQLLMQN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDELL 855-BAE96918 323QNITEEFYQSTCSAVSRGY 324 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAV (B)FSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYINNQLLPIVNQQSCRISNIETVIKETKCNGTDTKVKLIKQE IEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINLDKYKNAVTELQLLMQN DMPITNDQKKLMSSNVQIVRQQSYSIMSIMKEEVLAYVVQLP TPAANNRARRIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDELL 855-AFD34265 325QNITEEFYQSTCSAVSRGY 326 FLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAV (B)LSALRTGWYHCVITIELSN VSLSNGVSVCTIKVLDLKNYINNQLLPIVNQQSCRISNIETVIKETKCNGTDTKVKLIKQE IEFQQKNSRLLEIAREFSVNAGVTTPLSTYMLTNSELLSLINLDKYKNAVTELQLLMQN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLP TPAANNRARRIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSSEFDASISQVNEKINQSLAFIRRSDELL

TABLE G Sequence Index SEQ ID NO Description 1, 4, 81-210 Amino acidsequence of F0 precursor polypeptide of representative RSV subtype A 2,6, 211-263 Amino acid sequence of F0 precursor polypeptide ofrepresentative RSV subtype B 264-270 Amino acid sequence of F0 precursorpolypeptide of representative bovine RSV 3 Amino acid sequence of theectodomain (with foldon) of RSV A2, 5 Amino acid sequence of theectodomain (with foldon) of RSV A (Ontario) 7 Amino acid sequence of theectodomain (with foldon) of a RSV B strain  8-18 Nucleotide sequenceencoding the precursor polypeptide of representative RSV F mutants19-21, 32-39, Amino acid sequence of F precursor polypeptide ofrepresentative 271-278 RSV F mutants 22-31 Amino acid sequences of thelight chain variable domain and heavy chain variable domain of RSV Fantibodies 40  Amino acid sequence of T4 Fibritin foldon 41-80, 279-326Amino acid sequence of F2 polypeptide and F1 polypeptide ofrepresentative RSV F mutants

F. EXAMPLES

The invention is further described by the following illustrativeexamples. The examples do not limit the invention in any way. Theymerely serve to clarify the invention.

Example 1: Design and Preparation of RSV F Protein Mutants

1A: RSV F Mutants with Foldon Domain

This example illustrates the design and preparation of various RSV Fprotein mutants, which include a fibritin foldon trimerization domainand introduced amino acid mutations, such as engineered disulfide bondmutations, cavity-filling mutations, electrostatic mutations, or acombination thereof. Exemplary RSV F mutants, each of which isidentified by an unique identifier, such as pXCS501, pXCS601, etc., areprovided in Tables 1-6. Each of these mutants was designed and preparedbased on the amino acid sequence set forth in SEQ ID NO:3, which is alsoillustrated in FIG. 1. Amino acid residues 1-513 of the sequence of SEQID NO:3 are identical to amino acid residues 1-513 of the F0 precursorpolypeptide of native RSV A2 as set forth in SEQ ID NO:1, except for thethree naturally occurring substitutions, P102A, I379V and M447V, in thesequence of SEQ ID NO:3. Therefore, the amino acid sequences of theseexemplary F mutants are identical except for the introduced amino acidmutations as noted for each mutant listed in Tables 1-6. Each of theseRSV F protein mutants comprises two separate polypeptide chains. One ofthe polypeptide chains, the F2 polypeptide, comprises amino acids 26-109of SEQ ID NO:3 except for the introduced mutations as noted. The otherpolypeptide chain comprises the F1 polypeptide (residues 137-513) linkedto a foldon trimerization domain (residues 518-544) via a SAIG linker(residues 514-517). The signal peptide (residues 1-25) and pep27(residues 110-136) of SEQ ID NO:3 were cleaved from the F0 precursorduring the expression process. The process for expression andpurification of these exemplary RSV F mutants is described in Examples 2and 3.

1B: RSV F Mutants without Foldon Domain

RSV F mutant, pXCS899, which was devoid of foldon domain, was preparedin the same method described in Example 1A above, except that aminoacids 514-544 of the F0 precursor sequence of SEQ ID NO:3 were deleted.The amino acid sequence of the precursor polypeptide of pXCS899 is setforth in SEQ ID NO:271.

TABLE 1 Exemplary RSV F Protein Mutants Comprising Engineered DisulfideMutations Mutant ID Mutations pXCS501 I28C, G464C pXCS502 E30C, S466CpXCS503 Q34C, G471C pXCS504 S35C, G471C pXCS505 W52C, S150C pXCS506T54C, G151C pXCS507 S55C, L188C pXCS508 V56C, V187C pXCS509 V56C, T189CpXCS510 I57C, S190C pXCS511 T58C, K191C pXCS512 I59C, L193C pXCS513E60C, K196C pXCS514 L61C, L195C pXCS515 S62C, K196C pXCS516 S62C, I199CpXCS518 T103C, A147C pXCS519 T103C, I148C pXCS520 R106C, V144C pXCS521L138C, T337C pXCS522 G139C, P353C pXCS523 G139C, Q354C pXCS524 L142C,N371C pXCS525 G145C, M370C pXCS526 I148C, Y286C pXCS527 G151C, V300CpXCS528 G151C, Q302C pXCS529 V154C, V300C pXCS531 S155C, V300C pXCS532L158C, S290C pXCS534 V164C, K293C pXCS535 V164C, E294C pXCS536 T397C,P484C pXCS537 T397C, E487C pXCS538 K399C, S485C pXCS539 L410C, G464CpXCS540 L410C, S466C pXCS541 S443C, S466C pXCS542 L138C, P353C pXCS543G151C, I288C pXCS544 S155C, S290C pXCS545 S155C, S290C; I28C, G464CpXCS546 S155C, S290C, E30C, S466C pXCS547 S155C, S290C, Q34C, G471CpXCS548 S155C, S290C, S35C, G471C pXCS549 S155C, S290C, T397C, P484CpXCS550 S155C, S290C, T397C, E487C pXCS551 S155C, S290C, K399C, S485CpXCS553 S155C, S290C, L410C, S466C pXCS554 S155C, S290C, S443C, S466CpXCS556 R106C, V144C, S443C, S466C pXCS557 R106C, V144C, L142C, N371CpXCS558 R106C, V144C, T397C, P484C pXCS596 S55C, L188C, T103C, I148CpXCS597 S55C, L188C, R106C, V144C pXCS598 S55C, L188C, L142C, N371CpXCS599 S55C, L188C, T397C, P484C pXCS600 S55C, L188C, Q34C, G471CpXCS601 S55C, L188C, T397C, E487C pXCS602 S55C, L188C, S443C, S466CpXCS603 S55C, L188C, L410C, S466C pXCS604 S55C, L188C, S35C, G471CpXCS605 S55C, L188C, S62C, I199C pXCS606 T103C, I148C; Q34C, G471CpXCS607 T103C, I148C; S35C, G471C pXCS608 T103C, I148C; S62C, I199CpXCS609 T103C, I148C; L142C, N371C pXCS610 T103C, I148C; T397C, P484CpXCS611 T103C, I148C; T397C, E487C pXCS612 T103C, I148C; L410C, S466CpXCS613 T103C, I148C; S443C, S466C pXCS614 Q34C, G471C, S62C, I199CpXCS615 Q34C, G471C, R106C, V144C pXCS616 Q34C, G471C, L138C, T337CpXCS617 Q34C, G471C, L142C, N371C pXCS618 L142C, N371C, S35C, G471CpXCS619 L142C, N371C, S62C, I199C pXCS620 L142C, N371C, S155C, S290CpXCS621 L142C, N371C, T397C, P484C pXCS622 L142C, N371C, T397C, E487CpXCS623 L142C, N371C, L410C, S466C pXCS624 L142C, N371C, S443C, S466CpXCS625 R106C, V144C, S62C, I199C pXCS626 R106C, V144C, T397C, E487CpXCS627 R106C, V144C, L410C, S466C pXCS628 S55C, L188C, L138C, T337CpXCS629 S55C, L188C, G145C, M370C pXCS630 T103C, I148C; L138C, T337CpXCS712 S55C, L188C, R106C, V144C, L142C, N371C pXCS517 S62C, D200CpXCS530 S155C, I288C pXCS533 L158C, I291C pXCS552 S155C, S290C, L410C,G464C pXCS555 S155C, S290C, R106C, V144C

TABLE 2 Exemplary RSV F Protein Mutants Comprising Cavity FillingMutations Mutant ID Mutations pXCS559 S55I pXCS560 S55Y pXCS561 S62LpXCS562 S62Y pXCS563 S155H pXCS564 S155Y pXCS565 S190I pXCS566 S190MpXCS567 S190Y pXCS568 S290H pXCS569 S290M pXCS570 S290Y pXCS571 T54HpXCS572 T54I pXCS573 T58L pXCS574 T58M pXCS575 T189I pXCS577 T219IpXCS578 T219M pXCS579 T397I pXCS580 T397Y pXCS581 G151A pXCS582 G151HpXCS583 A147H pXCS584 A147I pXCS585 A298L pXCS586 A298M pXCS587 V164IpXCS588 V187I pXCS589 V192H pXCS590 V207I pXCS591 V220I pXCS592 V296IpXCS593 V300I pXCS594 V495Y pXCS595 R106W pXCS666 S190F, V207L pXCS691V495Y, S62L pXCS692 V495Y, T219M pXCS693 V495Y, T54H pXCS694 V495Y, T58LpXCS695 V495Y, V164I pXCS696 V495Y, V187I pXCS697 V495Y, V296I pXCS698V296I, S62L pXCS699 V296I, T219M pXCS700 V296I, T54H pXCS701 T54H, S62LpXCS702 T54H, T219M pXCS711 F488W pXCS576 T189Y

TABLE 3 Exemplary RSV F Protein Mutants Comprising ElectrostaticMutations Mutant ID Mutations pXCS631 E82Q pXCS632 E82S pXCS633 E82LpXCS634 E92D pXCS635 E92T pXCS636 E92Q pXCS637 E92F pXCS638 R106QpXCS639 R106N pXCS640 R106F pXCS641 K315F pXCS642 K315L pXCS643 K315IpXCS644 K315Q pXCS645 R339Q pXCS646 R339W pXCS647 R339F pXCS648 D392NpXCS649 D392S pXCS650 D392P pXCS651 K394M pXCS652 K394T pXCS653 K394FpXCS654 K399R pXCS655 K399M pXCS656 K399S pXCS657 D486H pXCS658 D486SpXCS659 D486T pXCS660 E487Q pXCS661 E487H pXCS662 E487D pXCS663 D489HpXCS664 D489S pXCS665 D489N

TABLE 4 Exemplary RSV F Protein Mutants Comprising Engineered DisulfideMutations and Cavity Filling Mutations Mutant ID Mutations pXCS667R106C-V144C; S443C-S466C; S55I pXCS668 R106C-V144C; L142C-N371C; S55IpXCS669 R106C-V144C; T397C-P484C; S55I pXCS670 R106C-V144C; S443C-S466C;T54H pXCS671 R106C-V144C; L142C-N371C; T54H pXCS672 R106C-V144C;T397C-P484C; T54H pXCS674 R106C-V144C L142C-N371C; T54H, S190Y pXCS679S62C-I199C; L142C-N371C; S55I pXCS680 S62C-I199C; L142C-N371C; T54HpXCS683 Q34C-G471C; L142C-N371C; S62L pXCS684 Q34C-G471C; L142C-N371C;T219M pXCS685 Q34C-G471C; L142C-N371C; T54H pXCS686 Q34C-G471C;L142C-N371C; V164I pXCS687 Q34C-G471C; L142C-N371C; V187I pXCS688Q34C-G471C; L142C-N371C; V296I pXCS689 Q34C-G471C; L142C-N371C; T397YpXCS690 Q34C-G471C; L142C-N371C; V495Y pXCS713 Q34C-G471C; S155C-S290C;T54H pXCS714 Q34C-G471C; S155C-S290C; V296I pXCS715 Q34C-G471C;S155C-S290C; V495Y pXCS716 Q34C-G471C; S155C-S290C; T54H, V495Y pXCS717Q34C-G471C; S155C-S290C; T54H, V296I pXCS718 Q34C-G471C; S155C-S290C;T54H, V296I, V495Y pXCS719 Q34C-G471C; S155C-S290C; S190I pXCS720S155C-S290C; L410C-S466C; T54H pXCS721 S155C-S290C; L410C-S466C; V296IpXCS722 S155C-S290C; L410C-S466C; V495Y pXCS723 S155C-S290C;L410C-S466C; T54H, V495Y pXCS724 S155C-S290C; L410C-S466C; T54H, V296IpXCS725 S155C-S290C; L410C-S466C; T54H, V296I, V495Y pXCS726S155C-S290C; L410C-S466C; S190I pXCS727 R106C-V144C; L142C-N371C; T54HpXCS728 R106C-V144C; L142C-N371C; V296I pXCS729 R106C-V144C;L142C-N371C; V495Y pXCS730 R106C-V144C; L142C-N371C; T54H, V495Y pXCS731R106C-V144C; L142C-N371C; T54H, V296I pXCS732 R106C-V144C; L142C-N371C;T54H, V296I, V495Y pXCS733 R106C-V144C; L142C-N371C; S190I pXCS734S55C-L188C; L142C-N371C; T54H pXCS735 S55C-L188C; L142C-N371C; V296IpXCS736 S55C-L188C; L142C-N371C; V495Y pXCS737 S55C-L188C; L142C-N371C;T54H, V495Y pXCS738 S55C-L188C; L142C-N371C; T54H, V296I pXCS739S55C-L188C; L142C-N371C; T54H, V296I, V495Y pXCS740 S55C-L188C;L142C-N371C; S190I pXCS741 Q34C-G471C; S55C-L188C; T54H pXCS742Q34C-G471C; S55C-L188C; V296I pXCS743 Q34C-G471C; S55C-L188C; V495YpXCS744 Q34C-G471C; S55C-L188C; T54H, V495Y pXCS745 Q34C-G471C;S55C-L188C; T54H, V296I pXCS746 Q34C-G471C; S55C-L188C; T54H, V296I,V495Y pXCS747 Q34C-G471C; S55C-L188C; S190I pXCS748 T103C-I148C; T54HpXCS749 T103C-I148C; V296I pXCS750 T103C-I148C; V495Y pXCS751T103C-I148C; T54H, V495Y pXCS752 T103C-I148C; T54H, V296I pXCS753T103C-I148C; T54H, V296I, V495Y pXCS754 T103C-I148C; S190I pXCS781S55C-L188C; T54H pXCS782 S55C-L188C; V296I pXCS783 S55C-L188C; V495YpXCS784 S55C-L188C; T54H, V495Y pXCS785 S55C-L188C; T54H, V296I pXCS786S55C-L188C; T54H, V296I, V495Y pXCS787 S55C-L188C; S190I pXCS789R106C-V144C; T54H pXCS790 R106C-V144C; V296I pXCS791 R106C-V144C; V495YpXCS792 R106C-V144C; T54H, V495Y pXCS793 R106C-V144C; T54H, V296IpXCS794 R106C-V144C; T54H, V296I, V495Y pXCS795 R106C-V144C; S190IpXCS797 L142C-N371C; T54H pXCS798 L142C-N371C; V296I pXCS799L142C-N371C; V495Y pXCS800 L142C-N371C; T54H, V495Y pXCS801 L142C-N371C;T54H, V296I pXCS802 L142C-N371C; T54H, V296I, V495Y pXCS803 L142C-N371C;S190I pXCS805 S155C-S290C; T54H pXCS806 S155C-S290C; V296I pXCS807S155C-S290C; V495Y pXCS808 S155C-S290C; T54H, V495Y pXCS809 S155C-S290C;T54H, V296I pXCS810 S155C-S290C; T54H, V296I, V495Y pXCS811 S155C-S290C;S190I pXCS812 Q34C-G471C; S155C-S290C; T54H, S190I pXCS815 S155C-S290C;L410C-S466C; I54H, S190I pXCS818 R106C-V144C; L142C-N371C; I54H, S190IpXCS821 S55C-L188C; L142C-N371C; I54H, S190I pXCS827 T103C-I148C; T54H,S190I pXCS828 T103C-I148C; S190I, V495Y pXCS830 S55C-L188C; T54H, S190IpXCS831 S55C-L188C; S190I, V495Y pXCS833 R106C-V144C; I54H, S190IpXCS834 R106C-V144C; S190I, V495Y pXCS836 L142C-N371C; T54H, S190IpXCS837 L142C-N371C; S190I, V495Y pXCS839 S155C-S290C; T54H, S190IpXCS840 S155C-S290C; S190I, V495Y pXCS889 T103C-I148C; S190I, V296IpXCS890 T103C-I148C; T54H, S190I, V296I pXCS891 S55C-L188C; S190I, V296IpXCS892 S55C-L188C; T54H, S190I, V296I pXCS893 R106C-V144C; S190I, V296IpXCS894 R106C-V144C; T54H, S190I, V296I pXCS895 L142C-N371C; S190I,V296I pXCS896 L142C-N371C; T54H, S190I, V296I pXCS897 S155C-S290C;S190I, V296I pXCS898 S155C-S290C; T54H, S190I, V296I

TABLE 5 Exemplary RSV F Protein Mutants Comprising Engineered DisulfideMutations and Electrostatic Mutations. Mutant ID Mutations pXCS755Q34C-G471C; S155C-S290C; D486S pXCS756 S155C-S290C; L410C-S466C; D486SpXCS757 R106C-V144C; L142C-N371C; D486S pXCS758 S55C-L188C; L142C-N371C;D486S pXCS759 Q34C-G471C; S55C-L188C; D486S pXCS760 T103C-I148C; D486SpXCS770 Q34C-G471C; S155C-S290C; D486S, E487Q pXCS771 Q34C-G471C;S155C-S290C; D486S, D489S pXCS772 Q34C-G471C; S155C-S290C; D486S, E487Q,D489S pXCS776 T103C-I148C; D486S, E487Q pXCS777 T103C-I148C; D486S,D489S pXCS778 T103C-I148C; D486S, E487Q, D489S pXCS779 T103C-I148C; E92DpXCS780 S55C-L188C; D486S pXCS788 R106C-V144C; D486S pXCS796L142C-N371C; D486S pXCS804 S155C-S290C; D486S pXCS883 S55C-L188C;L142C-N371C; D486S, E487Q pXCS884 S55C-L188C; L142C-N371C; D486S, D489SpXCS885 S55C-L188C; L142C-N371C; D486S, E487Q, D489S

TABLE 6 Exemplary RSV F Protein Mutants Comprising a Combination ofEngineered Disulfide Mutations, Cavity Filling Mutations, andElectrostatic Mutations. Mutant ID Mutations pXCS761 Q34C-G471C;S155C-S290C; T54H, D486S, E487Q, D489S, V495Y pXCS762 Q34C-G471C;S155C-S290C; T54H, V296I, D486S, E487Q, D489S pXCS763 Q34C-G471C;S155C-S290C; T54H, V296I, D486S, E487Q, D489S, V495Y pXCS764 Q34C-G471C;S55C-L188C; T54H, D486S, E487Q, D489S, V495Y pXCS765 Q34C-G471C;S55C-L188C; T54H, V296I, D486S, E487Q, D489S pXCS766 Q34C-G471C;S55C-L188C; T54H, V296I, D486S, E487Q, D489S, V495Y pXCS767 R106C-V144C;L142C-N371C; T54H, D486S, E487Q, D489S, V495Y pXCS768 R106C-V144C;L142C-N371C; T54H, V296I, D486S, E487Q, D489S pXCS769 R106C-V144C;L142C-N371C; T54H, V296I, D486S, E487Q, D489S, V495Y pXCS773T103C-I148C; T54H, D486S, E487Q, D489S, V495Y pXCS774 T103C-I148C; T54H,V296I, D486S, E487Q, D489S pXCS775 T103C-I148C; T54H, V296I, D486S,E487Q, D489S, V495Y pXCS842 T103C-I148C; T54H, S190I, D486S pXCS843T103C-I148C; S190I, D486S, V495Y pXCS844 T103C-I148C; T54H, S190I,D486S, V495Y pXCS845 T103C-I148C; T54H, D486S pXCS846 T103C-I148C;D486S, V495Y pXCS847 T103C-I148C; S190I, D486S pXCS848 T103C-I148C;V296I, D486S pXCS849 T103C-I148C; T54H, V296I, D486S pXCS850T103C-I148C; S190I, V296I, D486S pXCS851 T103C-I148C; T54H, S190I,V296I, D486S pXCS852 S55C-L188C; T54H, D486S pXCS853 S55C-L188C; S190I,D486S pXCS854 S55C-L188C; V296I, D486S pXCS855 S55C-L188C; T54H, S190I,D486S pXCS856 S55C-L188C; T54H, V296I, D486S pXCS857 S55C-L188C; S190I,V296I, D486S pXCS858 S55C-L188C; T54H, S190I, V296I, D486S pXCS859R106C-V144C; T54H, D486S pXCS860 R106C-V144C; S190I, D486S pXCS861R106C-V144C; V296I, D486S pXCS862 R106C-V144C; T54H, S190I, D486SpXCS863 R106C-V144C; T54H, V296I, D486S pXCS864 R106C-V144C; S190I,V296I, D486S pXCS865 R106C-V144C; T54H, S190I, V296I, D486S pXCS866L142C-N371C; T54H, D486S pXCS867 L142C-N371C; S190I, D486S pXCS868L142C-N371C; V296I, D486S pXCS869 L142C-N371C; T54H, S190I, D486SpXCS870 L142C-N371C; T54H, V296I, D486S pXCS871 L142C-N371C; S190I,V296I, D486S pXCS872 L142C-N371C; T54H, S190I, V296I, D486S pXCS873S155C-S290C; T54H, D486S pXCS874 S155C-S290C; S190I, D486S pXCS875S155C-S290C; V296I, D486S pXCS876 S155C-S290C; T54H, S190I, D486SpXCS877 S155C-S290C; T54H, V296I, D486S pXCS878 S155C-S290C; S190I,V296I, D486S pXCS879 S155C-S290C; T54H, S190I, V296I, D486S pXCS880S55C-L188C; L142C-N371C; T54H, S190I, D486S, E487Q, D489S pXCS881S55C-L188C; L142C-N371C; T54H, V296I, D486S, E487Q, D489S pXCS882S55C-L188C; L142C-N371C; T54H, S190I, V296I, D486S, E487Q, D489S pXCS886T103C-I148C; T54H, S190I, D486S, E487Q, D489S pXCS888 T103C-I148C; T54H,S190I, V296I, D486S, E487Q, D489S

Example 2. RSV F Mutant Expression Vector Construction

A nucleic acid molecule encoding the native RSV A2 F0 polypeptide setforth in SEQ ID NO:1 having the naturally-occurring substitutions P102A,I379V and M447V was mutated using standard molecular biology techniquesto encode a precursor polypeptide for a RSV F mutant having desiredintroduced amino acid mutations. The structure and components of theprecursor polypeptide are set forth in FIG. 1 and SEQ ID NO:3. Theprecursor polypeptide comprises a signal peptide (residues 1-25), F2polypeptide (residues 26-109), pep27 polypeptide (residues 110-136), F1polypeptide (residues 137-513), T4 fibritin foldon (residues 518-544),thrombin recognition sequence (547-552), purification tags (HIS-tag(residues 553-558)), Strep tag II (residues 561-568), and linkersequences (residues 514-517, 545, 546, 559, and 560).

The protein sequence of SEQ ID NO:3 was submitted for mammalian codonoptimization and synthesis by DNA2.0 (Menlo Park, Calif.). Thesynthesized gene product was introduced into a commercially availableexpression vector, pcDNA3.1/Zeo(+) (ThermoFisher Scientific, Waltham,Mass.) that had been modified to encode kanamycin resistance instead ofampicillin resistance and to encode the CAG promoter [Niwa, H.,Yamamura, K., & Miyazaki, J., Efficient selection for high-expressiontransfectants with a novel eukaryotic vector. Gene, 108(2), 193-199,1991] in place of the CMV promoter. Mutagenic oligonucleotides weredesigned with the QuikChange Primer Design algorithm (AgilentTechnologies, Santa Clara, Calif.), and all oligonucleotides werepurchased from Integrated DNA Technologies (Coralville, Iowa).Nucleotide substitutions, insertions, and deletions were incorporatedwith the QuikChange Lightning Multi Site-Directed Mutagenesis Kit(Agilent Technologies). Following digestion of the original plasmidtemplate with DpnI, the mutagenized F allele was re-amplified bypolymerase chain reaction (PCR) with high-fidelity Q5 DNA polymerase(New England Biolabs, Ipswich, Mass.) or PrimeSTAR HS (Premix) DNApolymerase (Takara/Clontech, Mountain View, Calif.), and the resultingproduct was inserted into a mammalian expression vector with theNEBuilder HiFi DNA Assembly Kit (New England Biolabs) or with GibsonAssembly Master Mix (New England Biolabs). The presence of the intendedsequence was confirmed by DNA sequencing. Plasmid DNA for transfectioninto Expi293 cells was purified with the QIAprep Spin MiniPrep Kit(Qiagen, Valencia, Calif.), or with the EndoFree Plasmid Mega Kit(Qiagen). For all commercial kits or reagents, procedures were performedaccording to the manufacturer's protocol.

Example 3. Expression and Purification of RSV F Protein Mutants

Protein for RSV F protein mutant evaluation was produced by transienttransfection of Expi293F cells (ThermoFisher, Waltham, Mass.) with DNAconstructs assembled and prepared as described in Example 2. Transienttransfections were carried out according to the manufacturer's protocol.

Clarified cell culture was concentrated 5-10 fold using tangential flowfiltration, followed by buffer exchange into a buffer suitable forcapture on a Ni-IMAC column. The conditioned cell culture mediumcontaining soluble F protein was loaded onto a Ni-IMAC column. Theproduct was eluted using increasing concentrations of imidazole. Thefractions containing product were pooled and then loaded on aStrep-Tactin column (IBA Life Sciences, Goettingen, Germany). Theproduct was eluted from the Strep-Tactin column using increasingconcentrations of desthiobiotin. Fractions containing product werepooled and dialysed into the final storage buffer. The crude culturesupernatants and purified proteins were used for in vitro and in vivoassays described herein.

Example 4: Stability of RSV F Protein Mutants

The stability of the designed RSV F protein mutants was evaluated bystress testing and storage stability experiments. During thermal stresstesting, crude culture supernatants of the designed mutants wereincubated for 1 hour at 50° C. or 60° C. and probed with the pre-fusionspecific monoclonal antibody D25 and the pre-fusion trimer-specificantibody AM14 in ELISA assays. The ratio of the antibody reactivity ofthe stressed versus unstressed sample is defined as the stressresistance parameter. More stable mutants are expected to have higherstress resistance. During storage stability assays, pre-fusion antibodyreactivity in crude culture supernatants after 1 week of storage at 4°C. was compared to the reactivity of the fresh culture supernatants. Theactivity ratio is defined as storage stability of the mutant.

Results are presented in Tables 7A-7C and 8A-8C. Stress resistance wascalculated as fractional pre-fusion specific mAb reactivity remainingafter stress (“NR”—No Reactivity was detected, “ND”—Not Determined). Themost stabilizing amino acid substitutions identified from screens of theindividual engineered disulfide mutants, cavity filling mutants andelectrostatic mutants (pre-fusion stability defined by D25 reactivityremaining after thermal stress) were combined into the combinationmutants. These combination mutants were also subjected to the thermalstress and probed with two monoclonal antibodies—D25(pre-fusion-specific) and AM14 (pre-fusion trimer-specific). Thepre-fusion trimer-specific quaternary epitope recognized by the AM14antibody is significantly more sensitive to thermal stress than the D25epitope (Table 8B). No significant AM14 reactivity was retained after60° C. stress by any of the combination mutants, yet most of the mutantsretained D25 reactivity after the 60° C. thermal stress. Thisobservation provides important evidence that the AM14 antibody is a muchmore precise indicator of pre-fusion structure loss, and particularlyloss of the pre-fusion trimeric state.

TABLE 7A Thermal and storage stability for mutants containing engineereddisulfides 50° C. stress 60° C. stress Mutant ID resistance, D25resistance, D25 Storage stability pXCS507 0.45 ± 0.09 <0.05  0.58pXCS519 1.07 ± 0.18 NR 0.75 pXCS524 0.64 ± 0.08 NR 1.00 pXCS544 0.52 ±0.04 NR 0.76 pXCS545 0.97 ± 0.12 0.52 ± 0.13 low expression pXCS546 1.11± 0.09 0.43 ± 0.09 low expression pXCS547 1.00 ± 0.04 0.49 ± 0.11 0.96pXCS548 1.04 ± 0.08 0.45 ± 0.10 low expression pXCS549 0.66 ± 0.09 0.24± 0.07 1.03 pXCS550 0.83 ± 0.02 0.19 ± 0.04 1.06 pXCS551 0.72 ± 0.08 NRlow expression pXCS553 1.12 ± 0.08 0.33 ± 0.03 1.31 pXCS554 2.08 ± 0.190.41 ± 0.08 low expression pXCS596 0.86 ± 0.09 0.02 0.80 pXCS597 0.50 ±0.05 0.05 1.07 pXCS598 0.75 ± 0.03 0.13 ± 0.03 1.09 pXCS599 0.68 ± 0.100.02 0.95 pXCS600 0.87 ± 0.09 0.15 ± 0.04 0.90 pXCS601 0.71 ± 0.08 0.040.57 pXCS602 0.75 ± 0.03 0.06 ± 0.01 0.58 pXCS603 0.67 ± 0.06 0.12 ±0.02 0.40 pXCS604 0.74 ± 0.03 ND low expression pXCS605 0.71 ± 0.04 0.16± 0.03 0.00 pXCS606 0.76 ± 0.06 NR low expression pXCS607 NR NR lowexpression pXCS608 0.62 ± 0.14 NR 0.00 pXCS609 0.76 ± 0.08 0.08 ± 0.010.28 pXCS610 0.34 ± 0.06 NR 0.00 pXCS611 0.35 ± 0.11 NR low expressionpXCS612 NR NR low expression pXCS613 0.3 NR 0.00 pXCS617 1.04 ± 0.040.43 ± 0.04 0.50 pXCS618 1.01 ± 0.07 0.30 ± 0.08 low expression pXCS6191.04 ± 0.08 0.34 ± 0.07 0.57 pXCS620 ND ND low expression pXCS621 0.87 ±0.03 0.14 ± 0.02 0.62 pXCS622 ND ND low expression pXCS623 0.91 ± 0.030.26 ± 0.03 low expression pXCS624 0.87 ± 0.06 0.18 ± 0.04 0.67 pXCS6280.83 ± 0.04 0.16 ± 0.02 0.71 pXCS629 1.01 ± 0.06 0.04 ± 0.03 1.00pXCS630 0.61 ± 0.04 NR 0.00

TABLE 7B Thermal and storage stability of mutants containing cavityfilling mutations 50° C. stress 60° C. stress Storage Mutant IDresistance, D25 resistance, D25 stability pXCS565 0.61 ± 0.06 0.03 ±0.01 0.74 pXCS571 0.46 ± 0.05 NR 0.81 pXCS592 0.38 ± 0.07 NR 0.025

TABLE 7C Thermal and storage stability of mutants containingelectrostatic mutations 50° C. stress 60° C. stress Storage Mutant IDresistance, D25 resistance, D25 stability pXCS658 1.05 ± 0.05 0.30 ±0.03 0.71 pXCS660 1.03 ± 0.03 NR 0.94 pXCS664 0.87 ± 0.03 NR 0.76

TABLE 8A Thermal and storage stability of mutants containing doublecombination mutations 50° C. stress 60° C. stress Mutant ID resistance,D25 resistance, D25 Storage stability pXCS674 0.73 ± 0.09 0.53 ± 0.071.47 pXCS683 1.10 ± 0.02 0.4  low expression pXCS684 1.05 ± 0.01 0.46 ±0.04 low expression pXCS685 1.10 ± 0.02 0.70 ± 0.06 low expressionpXCS686 1.17 ± 0.08 0.63 ± 0.05 low expression pXCS687 1.10 ± 0.04 0.50± 0.03 low expression pXCS688 1.09 ± 0.03 0.56 ± 0.08 low expressionpXCS689 1.06 ± 0.02 0.44 ± 0.07 low expression pXCS690 1.06 ± 0.06 0.50± 0.03 0.27 pXCS693 0.70 ± 0.05 0.08 ± 0.01 0.54 pXCS697 0.49 ± 0.040.06 low expression pXCS698 NR NR low expression pXCS699 0.31 ± 0.05 NRlow expression pXCS700 0.65 ± 0.05 0.03 0.67 pXCS701 0.36 NR lowexpression pXCS702 0.48 ± 0.02 NR low expression

TABLE 8B Thermal and storage stability for mutants containing triplecombination mutations 50° C. resistance, 60° C. stress Mutant ID AM14resistance, D25 Storage stability pXCS734 0.61 0.31 ± 0.00 1.09 ± 0.06pXCS735 0.70 0.37 ± 0.04 0.73 ± 0.13 pXCS738 0.72 0.37 0.58 ± 0.10pXCS740 0.69 0.41 ± 0.06 1.03 ± 0.11 pXCS749 ND 0.00 ± 0.10 0.65 ± 0.15pXCS752 ND 0.00 ± 0.10 0.62 ± 0.05 pXCS754 ND 0.00 ± 0.10 1.19 ± 0.06pXCS758 0.70 0.22 ± 0.04 1.32 ± 0.03 pXCS760 ND 0.82 ± 0.04 0.66 ± 0.22pXCS774 1.00 ± 0.16 0.74 ± 0.03 0.74 ± 0.10 pXCS776 1.40 ± 0.21 1.08 ±0.09 0.30 ± 0.16 pXCS777 1.03 ± 0.17 1.17 ± 0.05 0.54 ± 0.21 pXCS7781.14 ± 0.18 0.36 ± 0.07 0.34 ± 0.06 pXCS779 0.85 ± 0.15 0.00 ± 0.00 0.51± 0.08 pXCS780 0.70 ± 0.17 0.11 ± 0.00 0.82 ± 0.08 pXCS781 0.88 ± 0.170.00 ± 0.10 0.94 ± 0.05 pXCS782 0.55 ± 0.18 0.07 ± 0.01 0.67 ± 0.13pXCS785 1.01 ± 0.18 0.00 ± 0.10 1.08 ± 0.18 pXCS787 0.82 ± 0.17 0.14 ±0.01 0.82 ± 0.19 pXCS804 0.79 ± 0.11 0.19 ± 0.01 0.98 ± 0.08 pXCS8050.72 ± 0.15 0.24 ± 0.01 0.78 ± 0.24 pXCS806 0.40 ± 0.13 0.16 ± 0.03 0.79± 0.13 pXCS809 0.84 ± 0.12 0.29 ± 0.04 0.82 ± 0.11 pXCS811 0.67 ± 0.100.30 ± 0.04 1.03 ± 0.16 pXCS827 0.88 ± 0.07 0.06 0.50 ± 0.10 pXCS8301.01 ± 0.15 0.14 ± 0.02 0.53 ± 0.10 pXCS839 0.82 ± 0.06 0.55 ± 0.03 0.57± 0.14 pXCS842 0.87 ± 0.14 0.88 ± 0.02 0.57 ± 0.10 pXCS845 1.00 ± 0.111.11 ± 0.11 0.50 ± 0.20 pXCS847 0.92 ± 0.14 0.74 ± 0.01 0.54 ± 0.11pXCS848 1.24 ± 0.12 1.00 ± 0.03 0.15 ± 0.29 pXCS849 0.92 ± 0.30 1.08 ±0.10 0.59 ± 0.14 pXCS850 0.88 ± 0.22 0.70 ± 0.01 0.75 ± 0.16 pXCS8510.95 ± 0.09 0.84 ± 0.05 0.79 ± 0.10 pXCS852 0.86 ± 0.13 0.78 ± 0.02 0.89± 0.03 pXCS853 0.98 ± 0.10 0.10 ± 0.01 0.53 ± 0.11 pXCS854 0.93 ± 0.080.08 ± 0.01 0.55 ± 0.14 pXCS855 0.94 ± 0.10 0.81 ± 0.07 0.53 ± 0.10pXCS856 0.97 ± 0.10 0.78 ± 0.00 0.59 ± 0.12 pXCS857 0.90 ± 0.14 0.110.91 ± 0.07 pXCS858 0.95 ± 0.10 0.78 ± 0.08 0.94 ± 0.06 pXCS873 0.77 ±0.22 1.11 ± 0.01 0.36 ± 0.13 pXCS874 0.93 ± 0.20 0.46 ± 0.01 0.78 ± 0.19pXCS875 0.62 ± 0.16 0.23 ± 0.00 0.50 ± 0.10 pXCS876 0.90 ± 0.20 1.06 ±0.04 0.52 ± 0.10 pXCS877 0.49 ± 0.20 1.09 ± 0.00 0.41 ± 0.09 pXCS8780.66 ± 0.16 0.40 ± 0.04 0.47 ± 0.16 pXCS879 0.82 ± 0.20 0.84 ± 0.04 0.50± 0.20 pXCS880 0.68 ± 0.20 0.87 ± 0.07 0.46 ± 0.15 pXCS881 0.68 ± 0.230.92 ± 0.03 0.47 ± 0.26 pXCS882 0.75 ± 0.21 0.94 ± 0.01 0.44 ± 0.16pXCS883 0.81 ± 0.13 0.40 ± 0.01 0.47 ± 0.24 pXCS884 0.69 ± 0.15 0.43 ±0.05 0.45 ± 0.17 pXCS885 0.60 ± 0.21 0.47 ± 0.01 0.45 ± 0.13 pXCS8860.89 ± 0.13 0.70 ± 0.02 0.45 ± 0.14 pXCS888 0.86 ± 0.14 0.81 ± 0.05 0.43± 0.10 pXCS889 0.99 ± 0.14 0.00 ± 0.10 0.86 ± 0.10 pXCS890 0.72 ± 0.340.10 ± 0.03 1.08 ± 0.05 pXCS891 0.93 ± 0.06 0.08 ± 0.01 1.08 ± 0.06pXCS892 0.95 ± 0.13 0.18 ± 0.01 1.09 ± 0.04 pXCS897 0.60 ± 0.28 0.42 ±0.04 0.67 ± 0.37 pXCS898 0.94 ± 0.46 0.48 ± 0.05 0.81 ± 0.19 DS-Cav10.60 0.22 0.90

TABLE 8C Thermal stability of a mutant devoid of a foldon trimerizationdomain (pXCS899) 50° C. 60° C. stress resistance, resistance, 50° C.resistance, 60° C. stress Mutant ID AM14 AM14 D25 resistance, D25pXCS899 0.684 0.323 0.906 0.287

Example 5: Conformational Integrity of RSV F Protein Mutants Evaluatedwith a Panel of Monoclonal Antibodies

The purpose of the study was to identify RSV F protein mutants thatmaintain the structural integrity of a RSV-F pre-fusion conformation,including a pre-fusion trimer conformation and association. Each mutantwas tested against a panel of reference mAbs that includes two site ø-and pre-fusion-specific mAbs (AM22 and D25), one mAb that binds anepitope close to site II is also pre-fusion-specific (MPE8), one siteII-specific mAb that binds both pre-fusion and post-fusion F(palivizumab, Synagis®), one pre-fusion trimer-specific mAb (AM14) and asite IV-specific antibody that binds both pre-fusion and post-fusion F(101F). RSV F protein mutants maintained in a pre-fusion conformationwere expected to bind all the reference antibodies tested.

The OCTET HTX (ForteBio, Pall Corporation, Port Washington, N.Y.)instrument, which measures kinetics of real-time biomolecularinteractions, was used to evaluate the antibody reactivity for eachmutant. Experiments were conducted with 1000 rpm agitation, at 30° C.temperature in 96-well black plates (Greiner Bio-One, Monroe, N.C.) witha final volume of 200 μL per well. Anti-HIS biosensor tips wereequilibrated in phosphate-buffered saline (PBS), 2% bovine serum albumin(BSA), 0.05% Tween 20 (PBT) for 10 min before commencing bindingmeasurements. HIS-tagged mutant proteins were captured by anti-HISbiosensors for 5 min. Baseline was established in PBT for 3 min beforethe association step with 20 nM antibodies in PBT for 10 min.Dissociation of all antibodies was allowed for 20 min in the same wellsused to establish baseline. OCTET data analysis software (version 8.2,Pall Corp.) was used for kinetic analysis, based on curve fitting ofassociation and dissociation steps, and assuming 1:1 reversible bindinginteraction. Binding responses (nm shift) for each of the mutants withvarious reference monoclonal antibodies are presented in Tables 9A and9B. A response value <0.10 was considered negative and is the limit ofdetection (LOD) for this assay. Values ≥0.10 were considered positiveand indicated antibody binding to individual mutants. Varying degrees ofbinding were observed across the mutants and with each antibody.However, the majority of combination mutants were bound by 101F andSynagis whereas mutants such as pXCS735 and pXCS776 for example showedloss of binding to at least one pre-fusion-specific mAb. Loss of bindingindicated lack of an intact pre-fusion conformation.

TABLE 9A OCTET Results for Pre-Fusion-Specific Antibodies Response (nmshift) Mutant ID AM22 D25 MPE8 AM14 pXCS734 0.284 0.590 0.818 0.931pXCS735 LoD 0.302 0.375 0.442 pXCS738 0.273 0.545 0.741 0.899 pXCS7400.172 0.422 0.546 0.524 pXCS749 LoD 0.153 0.206 0.178 pXCS752 0.2340.463 0.703 0.641 pXCS754 0.298 0.562 0.676 0.816 pXCS758 0.273 0.5890.816 0.772 pXCS760 0.121 0.176 0.151 0.203 pXCS774 0.340 0.595 0.7950.738 pXCS776 LoD LoD 0.127 LoD pXCS777 0.121 0.201 0.270 0.285 pXCS778LoD LoD LoD 0.121 pXCS779 0.125 0.200 0.232 0.329 pXCS780 0.290 0.4950.608 0.684 pXCS781 0.423 0.666 0.876 1.053 pXCS782 0.222 0.378 0.5130.580 pXCS785 0.465 0.727 0.937 0.955 pXCS787 0.225 0.464 0.621 0.445pXCS804 0.267 0.432 0.605 0.522 pXCS805 0.173 0.282 0.443 0.355 pXCS8060.152 0.252 0.347 0.330 pXCS809 0.185 0.281 0.392 0.444 pXCS811 0.3220.490 0.638 0.714 pXCS827 0.450 0.692 0.880 0.881 pXCS830 0.465 0.7070.923 0.878 pXCS839 0.390 0.593 0.782 0.731 pXCS842 0.493 0.780 0.9320.930 pXCS845 0.314 0.495 0.699 0.653 pXCS847 0.484 0.732 0.871 0.935pXCS848 0.109 0.188 0.266 0.235 pXCS849 0.430 0.668 0.908 0.886 pXCS8500.517 0.839 1.038 1.018 pXCS851 0.508 0.824 1.025 1.027 pXCS852 0.5650.881 1.126 1.144 pXCS853 0.484 0.746 0.905 0.883 pXCS854 0.453 0.6930.886 0.884 pXCS855 0.523 0.778 0.982 0.992 pXCS856 0.568 0.827 1.0631.094 pXCS857 0.563 0.890 1.091 1.110 pXCS858 0.547 0.840 1.061 1.097pXCS873 0.192 0.398 0.537 0.434 pXCS874 0.444 0.681 0.851 0.771 pXCS8750.205 0.459 0.623 0.540 pXCS876 0.268 0.523 0.702 0.600 pXCS877 0.2130.419 0.573 0.525 pXCS878 0.324 0.666 0.812 0.833 pXCS879 0.351 0.6800.826 0.804 pXCS880 0.213 0.563 0.684 0.505 pXCS881 0.178 0.556 0.7630.623 pXCS882 0.219 0.516 0.691 0.463 pXCS883 0.233 0.553 0.704 0.484pXCS884 0.323 0.576 0.784 0.714 pXCS885 0.105 0.327 0.408 0.239 pXCS8860.443 0.715 0.863 0.872 pXCS888 0.434 0.726 0.878 0.875 pXCS889 0.4770.720 0.889 0.870 pXCS890 0.538 0.778 0.999 0.994 pXCS891 0.461 0.7190.920 0.828 pXCS892 0.542 0.756 1.032 1.000 pXCS897 0.406 0.605 0.7450.740 pXCS898 0.416 0.614 0.816 0.795 DS Cav1 0.469 0.714 0.810 0.842Note: LoD = limit of detection, ND = not determined

TABLE 9B OCTET Results for Antibodies 101F and Synagis Response (nmshift) Mutant ID 101F Synagis pXCS734 0.843 0.653 pXCS735 0.813 0.560pXCS738 0.774 0.519 pXCS740 0.759 0.611 pXCS749 0.629 0.480 pXCS7520.739 0.520 pXCS754 0.665 0.394 pXCS758 0.743 0.431 pXCS760 0.345 0.334pXCS774 0.742 0.530 pXCS776 0.139 0.123 pXCS777 0.389 0.329 pXCS7780.118 0.124 pXCS779 0.340 0.286 pXCS780 0.623 0.471 pXCS781 0.786 0.536pXCS782 0.615 0.468 pXCS785 0.763 0.580 pXCS787 0.615 0.547 pXCS8040.788 0.574 pXCS805 0.810 0.598 pXCS806 0.865 0.621 pXCS809 0.687 0.554pXCS811 0.768 0.606 pXCS827 0.973 0.898 pXCS830 0.901 0.839 pXCS8390.900 0.880 pXCS842 0.942 0.853 pXCS845 0.798 0.782 pXCS847 0.941 0.960pXCS848 0.400 0.394 pXCS849 0.999 0.991 pXCS850 1.040 1.076 pXCS8510.991 1.002 pXCS852 1.072 1.014 pXCS853 0.842 0.878 pXCS854 0.851 0.857pXCS855 0.914 0.894 pXCS856 0.957 0.935 pXCS857 1.016 1.056 pXCS8580.981 1.010 pXCS873 0.809 0.798 pXCS874 0.881 0.844 pXCS875 0.912 0.833pXCS876 0.820 0.727 pXCS877 0.842 0.823 pXCS878 0.832 0.776 pXCS8790.838 0.725 pXCS880 0.693 0.735 pXCS881 0.812 0.786 pXCS882 0.651 0.714pXCS883 0.719 0.807 pXCS884 0.798 0.792 pXCS885 0.666 0.737 pXCS8860.807 0.853 pXCS888 0.839 0.873 pXCS889 1.020 0.946 pXCS890 0.999 0.949pXCS891 0.919 0.851 pXCS892 0.960 0.889 pXCS897 0.939 0.901 pXCS8980.802 0.773 DS Cav1 0.821 0.704

Example 6. Molecular Weight and Size Distribution Analysis of SelectedPre-Fusion RSV F Mutants

Stabilized pre-fusion F mutants were analyzed by SDS-PAGE followed bywestern blotting with the RSV F-specific monoclonal antibody L4 [Walsh EE, Cote P T, Fernie B F et al. Analysis of the Respiratory SyncytialVirus Fusion Protein Using Monoclonal and Polyclonal Antibodies. J. Gen.Virol. 76: 505-513, 1986.]. FIG. 2A shows SDS-PAGE mobility profiles forrepresentative mutants pXCS847, pXCS851, pXCS852, and DS-Cav1. In allcases a major band with an apparent molecular weight between 55 and 60kDa, as expected for the monomeric RSV F mutants, was present undernon-reducing conditions. The observed slight change in mobility betweenDS-Cav1 and mutants pXCS847, pXCS851, and pXCS852 could be due to thenature of the individual disulfide bonds and the resulting effect on theoverall compactness of the protein in the unfolded state andaccessibility to SDS.

FIG. 2B describes molecular weights and size distributions of themutants pXCS847, pXCS851, pXCS852, and DS-Cav1 in solution under nativeconditions. The molecular weights and size distributions were estimatedfrom sedimentation velocity analysis using the analyticalultracentrifuge. Purified protein was centrifuged at 35,000 rpm, at 20°C., and UV absorbance across the sample cells was monitored at 280 nm.Data were fit to the continuous c(s) distributions, assuming the samefrictional ratio for all of the sedimenting species in the cell. Allproteins sedimented with sedimentation coefficient of ˜7.6 S andapparent molecular weight of ˜180 kDa, indicating that purified proteinsare trimeric in solution. The expected molecular weight of the RSV Ftrimer, calculated from its amino acid composition is 171 kDa.

Example 7. Circular Dichroism Spectroscopy to Characterize Secondary andTertiary Structure Integrity of the Designed RSV F Protein Mutants

Both far- and near-UV CD spectra were recorded on a Jasco J-810automated recording spectropolarimeter, equipped with a Peltier-type6-position temperature-controlled cell holder. Far-UV CD spectra wererecorded at 0.10-0.12 mg/ml protein concentration in 1×PBS, pH 7.4, in 1mm rectangular quartz cells between 200 and 260 nm every 0.1 nm at 100nm/min, with 3 nm band width. Five spectra were collected and averagedfor each sample. Near-UV CD spectra were recorded at 0.4-0.5 mg/mlprotein concentration in 1×PBS, pH 7.4, in 1 cm rectangular quartz cellsbetween 250 and 320 nm every 0.1 nm at 100 nm/min, with 3 nm band width.Five spectra were collected and averaged for each sample as well. Datawere corrected for the buffer baseline contributions and normalized toeither mean residue ellipticity (far-UV CD) or molar ellipticity(near-UV CD), using established relationships.

The results are shown in FIGS. 3A and 3B. Both far- and near-UV CD datashow that all proteins retain well defined secondary and tertiarystructure. Furthermore, obvious similarity of the far- and near-UV CDspectra indicates that overall secondary and tertiary structures of themutants are similar, and structural integrity of the proteins ispreserved.

Example 8. Structural Stability of the Designed RSV F Protein Mutants

The structural stability of the purified RSV F protein mutants wascharacterized using differential scanning calorimetry (DSC). DSCexperiments were conducted on a VP-DSC microcalorimeter (MicroCal,Northampton, Mass.). Protein concentration was determinedspectrophotometrically and corrected for light scattering contribution.Protein samples in 1×PBS, pH 7.4 at 0.2-0.5 mg/mL (1.0-2.4 micromolartrimer concentration) were scanned from 10° C. to 80° C. at 90° C./hr,with a response time of 8 seconds and pre-scan equilibration time of 5minutes. Depending on the number of the observed transitions inthermograms, heat capacity profiles were fit to the 2- or 3-stateunfolding models using Origin 7.0 software provided by the DSCmanufacturer. Melting temperatures of the first observable transitionsare given as melting temperatures of each mutant.

DSC data show almost all of the designed mutants are more stable thanDS-Cav1 (Table 10). Melting temperatures (defined as DSC maxima of thefirst observable DSC peak in each experiment, Table 10) of all mutants(with the exception of pXCS738) are higher than DS-Cav1 by up to 18° C.DSC data show that computational protein design described in Example 1succeeded in producing significantly more stable RSV F mutants that alsoretain a pre-fusion conformation (Octet data, Example 5).

TABLE 10 Melting temperatures of RSV F protein mutants. Meltingtemperatures were calculated from the DSC experiments (as described inExample 8). Mutant ID T_(m1), ° C. DS-Cav1 52.9 ± 0.0 pXCS738 52.5 ± 0.1pXCS780 65.2 ± 0.0 pXCS830 58.3 ± 0.0 pXCS847 68.4 ± 0.0 pXCS851 70.4 ±0.0 pXCS852 69.2 ± 0.0 pXCS853 65.2 ± 0.0 pXCS855 69.3 ± 0.0 pXCS87454.8 ± 1.0 pXCS881 70.6 ± 0.0 pXCS898 59.6 ± 0.5

Example 9. Mechanism of the Pre-Fusion Trimer Conformation Loss

In order to characterize a specific structural pathway leading to lossof the pre-fusion conformation, we subjected purified DS-Cav1 to thermalstress testing. The purified glycoprotein (0.5 mg/ml in 1×PBS, pH 7.4)was incubated at 50° C. and 60° C. for 30, 60 and 120 minutes. Bindingof the pre-fusion-specific mAb D25 and the pre-fusion trimer-specificmAb AM14 to the stressed protein was assessed via ELISA experiments asdescribed in Example 4). The structural integrity of the protein wascharacterized via CD and DSC as described in Examples 7 and 8,respectively. The results are shown in FIGS. 4-6.

The relative AM14 and D25 reactivities of the stressed samples are shownin FIG. 4. Both D25 and AM14 reactivity of DS-Cav1 remain largelyunchanged after up to 2 hours of incubation at 50° C. In contrast,reactivity to both pre-fusion specific antibodies is progressively lostduring 60° C. treatment. Furthermore, AM14 reactivity is lost morequickly than D25 reactivity, indicating that the quaternary pre-fusionAM14 epitope is disrupted earlier than the D25 epitope. The resulthighlights an advantage of the AM14 antibody as a probe for thedetection of the pre-fusion trimer conformation loss.

DSC assessment of the unstressed DS-Cav1 (FIG. 5) shows that the proteinundergoes a reversible conformational transition between 50° C. and 60°C. This transition does not correspond to the loss of the pre-fusionconformation, which is irreversible. Furthermore, this transition doesnot result from the global unfolding of the protein as DS-Cav1 retainsdefined far- and near-UV CD spectra (FIGS. 6A and 6B), indicating thatthe protein remains folded under these conditions. The most likelyexplanation of the observed DSC transition is reversible loss of thequaternary structure of the protein, i.e., at least local dissociationof the pre-fusion trimer. This dissociation is required for the initialsteps toward loss of the pre-fusion conformation, since neither AM14 norD25 reactivity is appreciably lost before that transition takes place(FIG. 4, ELISA data). These data emphasize the importance of trimerintegrity for the stability of the pre-fusion conformation. Trimerintegrity can only be confirmed by the reactivity against quaternaryepitope-specific antibody AM14, but not the site 0 specific antibodyD25.

These data suggest that the loss of the pre-fusion conformation occursvia the following pathway:N ₃↔3N→3U→U _(n)Native trimer (N₃) reversibly dissociates into native monomers (3N),which slowly and irreversibly lose pre-fusion conformation (3U) andultimately aggregate, forming high molecular mass species (U_(n)). Thetrimer dissociation, in turn, means that DS-Cav1 will display proteinconcentration-dependent resistance against thermal stress: a decrease intotal protein concentration will promote trimer dissociation, which, inturn, will accelerate pre-fusion conformation loss. In contrast,stabilized pre-fusion F mutants (e.g. 851) should show little to noconcentration dependence of their stress resistance, provided they weremade sufficiently stable. FIGS. 7A and 7B provide further experimentalevidence to support this hypothesis. Protein samples were seriallydiluted and subjected to 50° C. stress for one hour. AM14 and D25reactivities remaining after stress in relation to the control(unstressed) samples were assessed in ELISA assays. Stress resistance ofDS-Cav1 shows pronounced dependence on protein concentration, asdetermined by either AM14 (FIG. 7A) or D25 (FIG. 7B) antibodyreactivity. In contrast, stress resistance of the stabilized mutantspXCS851 and pXCS898 remains largely unchanged over the same proteinconcentration range.

Example 10: Stabilized RSV F Protein Mutants in Pre-Fusion ConformationElicit Neutralizing Antibody Responses in Mice

Female Balb/c mice were immunized with either 0.025 μg or 0.25 μg ofeither DS-Cav1, wild-type F, F mutants pXCS738, pXCS780, pXCS830,pXCS847, pXCS851, pXCS852, pXCS853, pXCS855, pXCS874, pXCS881, orpXCS898, or, with or without 0.1 mg per dose aluminum phosphate (AlPO4)as adjuvant. Immunizations were given intramuscularly at weeks 0 and 3(Table 11). Pre (week 0) and post-dose 2 (PD2, week 5) sera wereevaluated in an RSV subfamily A neutralization assay as described withminor modifications [Eyles J E, Johnson J E, Megati S, et al.Nonreplicating vaccines can protect african green monkeys from theMemphis 37 strain of respiratory syncytial virus. J Inf Dis.208(2):319-29, 2013.]. Briefly, neutralizing antibody titers weredetermined as the serum dilution factor resulting in a 50% reduction ininfectious units. Results are reported as the geometric mean titer from10 mice per group. Sera with no detectable virus neutralization wereassigned a titer of 20. Fold rise in geometric mean titers are reportedas the ratio of post-dose 2 (PD2) to pre-immunization titers within eachgroup.

TABLE 11 Immunization schedule of the murine immunogenicity studycomparing pre-fusion F mutants. Pre-fusion F Ag 0.025 μg and 0.25 μgwith and without AlPO4 dose (0.1 mg/mL) Vaccination Weeks 0, 3 BleedWeeks 0 (Pre) 3 (PD1) 5 (PD2)

All mutants tested elicited a neutralizing antibody response followingtwo immunizations in mice (Table 12). Overall, antibody titers wereconsistently higher at both antigen doses for mutants, pXCS830, pXCS847,pXCS851, and pXCS852, demonstrating that these mutants were the moreimmunogenic forms of a stabilized RSV pre-fusion F glycoprotein (Table12 and 13, FIG. 8). Comparison of the PD2 50% neutralizing antibodytiters with their corresponding mutants' in vitro characterization datashows correlation between the PD2 neutralizing antibody titer and theAM14 thermal stress resistance (FIG. 9). This result suggests that AM14binding, which is specific for the pre-fusion trimeric state, correlateswith the mutants' immunogenicity.

TABLE 12 Geometric mean neutralizing antibody titers of Balb/c micefollowing immunization with RSV F mutants. 0.025 μg + 0.25 μg + 0.025 μgNo 0.25 μg No AIPO4 AIPO4 adjuvant adjuvant Mutant ID Pre PD2 Pre PD2Pre PD2 Pre PD2 pXCS738 20 72 20 632 20 21 20 27 pXCS780 20 2373 20 131120 59 20 108 pXCS830 20 2615 20 3219 20 45 20 265 pXCS847 20 ND 20 ND 20129 20 518 pXCS851 20 1275 20 4393 20 59 20 237 pXCS852 20 1388 20 510020 135 20 331 pXCS853 20 690 20 1225 20 69 20 535 pXCS855 20 2004 201232 20 49 20 156 pXCS874 20 77 20 2929 20 34 20 86 pXCS881 20 53 202391 20 20 20 36 pXCS898 20 427 20 2642 20 39 20 87 DS-Cav1 20 271 202319 20 23 20 87 Wild type F 20 326 20 948 20 23 20 50 ND, not done.

TABLE 13 Fold rise in neutralizing antibody titers of Balb/c micefollowing immunization with RSV F mutants. 0.025 mg + 0.25 mg + 0.025 mgNo 0.25 mg No AlPO4 AlPO4 adjuvant adjuvant pXCS738 3.6 31.6 1.1 1.4pXCS780 118.7 65.6 3.0 5.4 pXCS830 130.8 161.0 2.3 13.3 pXCS847 N/A N/A6.5 25.9 pXCS851 63.8 219.7 3.0 11.9 pXCS852 69.4 255.0 6.8 16.6 pXCS85334.5 61.3 3.5 26.8 pXCS855 100.2 61.6 2.5 7.8 pXCS874 3.9 146.5 1.7 4.3pXCS881 2.7 119.6 1.0 1.8 pXCS898 21.4 132.1 2.0 4.4 DS-Cav1 13.6 116.01.2 4.4 Wild type F 16.3 47.4 1.2 2.5 N/A, not available.

Example 11. RSV F Mutants Comprising Introduced Cysteine Mutations inthe HRB Region

11A. Preparation of RSV F Mutants Comprising Introduced Mutations in theHRB Region

Representative RSV F mutants that comprise introduced cysteine mutationsin the HRB region (approximately amino acids 476-524 of the F0polypeptide) are provided in Table 14, where the specific mutations inthis region in each mutant are noted. In addition to the mutations inthe HRB region, each of these mutants also includes introduced mutationsS55C, L188C, T54H, and D486S. These mutants were prepared by methodssimilar to those described in Examples 1-3. In brief, a precursorpolypeptide consisting of 545 amino acids was prepared for each mutant,which comprises: (1) amino acids 1-529 of the sequence of SEQ ID NO:1except for a deletion of 41 amino acids between residues 104 and 144;(2) the introduced mutations (S55C, L188C, T54H, and D486S) outside ofthe HRB region, (3) a thrombin protease recognition sequence; (4) afoldon domain; (5) a HIS-tag; (6) a Streptag II; (7) linker sequences;and (8) the introduced cysteine mutations as noted. The signal peptide,which comprises amino acids 1-25, was cleaved from the precursor duringthe expression process. The foldon domain was also cleaved from themutants, which was achieved by digestion with 500 ug/ml bovinealpha-thrombin (HTI) overnight at room temperature after the expressionprocess.

TABLE 14 Exemplary RSV F protein mutants comprising engineered disulfidemutations in the HRB region SEQ ID NO of Amino Acid Sequence Mutant IDMutations in HRB Region of Precursor Polypeptide pXCS1106 K508C, S509C272 pXCS1107 N515C, V516C 273 pXCS1108 T522C, T523C 274 pXCS1109 K508C,S509C, N515C, V516C 275 pXCS1110 K508C, S509C, T522C, T523C 276 pXCS1111N515C, V516C, T522C, T523C 277 pXCS1112 K508C, S509C, N515C, V516C, 278T522C, T523C

11B. Stability of RSV F Mutants Comprising Introduced Cysteine Mutationsin the HRB Region

Stability of RSV F Mutants provided in Table 14 was assessed accordingto the method described in Example 8 and Example 4. For thermalstability assessment for mutant pXCS1106, purified pXCS1106 protein,from which the foldon had been cleaved, was diluted into conditionedmedium at a concentration of 12 μg/mL. Stress resistance for pXC1106 wascalculated as fractional pre-fusion specific mAb reactivity remainingafter stress. Results are presented in Tables 15 and 16, respectively.

TABLE 15 Melting temperatures of RSV F protein mutants Mutant ID T_(m1),° C. pXCS1106 66.3 pXCS1108 66.7 pXCS1109 66.5 pXCS1110 67.0 pXCS111166.5 pXCS1112 67.2

TABLE 16 Thermal stability of RSV F Mutant pXCS1106 50° C. 60° C. stress50° C. 60° C. stress resistance, resistance, resistance, resistance,Mutant ID AM14 AM14 D25 D25 pXCS1106 1.041 0.720 1.080 1.019

Listing of Raw SequencesSEQ ID NO: 1. Amino Acid Sequence of the Full Length F0 of Native RSVA2(GenBank GI: 138251; Swiss Prot P03420)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGVVYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNSEQ ID NO: 2. Amino Acid Sequence of the Full Length F0 of Native RSVB(18537 strain; GenBank GI: 138250; Swiss Prot P13843)MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGVVYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGVVYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK SEQ ID NO: 3. RSV A2 F Ectodomain with foldonMELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGVVYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEKSEQ ID NO: 4: RSV RSVA/Homo sapiens/USA/LA2_21/2013 F(Ontario) NativeAmino Acid Sequence (GenBank GI: AHX57185):MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNSEQ ID NO: 5: RSVRSVA/Homo sapiens/USA/LA2_21/2013 F Ectodomain with foldon:MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEKSEQ ID NO: 6: RSVRSVB/Homo sapiens/PER/FPP00592/2011 F (Buenos Aires)Native Amino Acid Sequence (GenBank GI: AHV80758):MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGVVYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSKSEQ ID NO: 7: RSV RSVB/Homo sapiens/PER/FPP00592/2011 F Ectodomain with foldon:MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGVVYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEKSEQ ID NO: 8: Nucleotide Sequence Encoding Pre-cursor Polypeptide of pXCS738:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccactgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgtgtggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacatccaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaaatcctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgtgtagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttccgacgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680;P102A  (naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; T54H: 160-162; S55C:163-165; L1420: 424-426; L188C: 562-564; V2961: 886-888; N3710: 1111-1113]SEQ ID NO: 9: Nucleotide Sequence Encoding Precursor Polypeptide of pXCS780:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtacacctgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacatccaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; S55C: 163-165; L188C:562-564; D486S: 1456-1458]SEQ ID NO: 10: Nucleotide Sequence Nucleotide Sequence Encoding Pre-coursor Polypeptide ofpXCS830:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccactgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttccgacgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; T54H: 160-162; S55C:163-165; L188C: 562-564; S190I: 568-570]SEQ ID NO: 11: Nucleotide Sequence Nucleotide Sequence Encoding Pre-coursor Polypeptideof pXCS847:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtacaccagcgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgcctgtaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcatgtgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgctgacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; T1030: 307-309; 11480:442-444; S190I: 568-570; D486S: 1456-1458]SEQ ID NO: 12: Nucleotide Sequence Nucleotide Sequence Encoding Pre-coursor Polypeptideof pXCS851:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccacagcgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgcctgtaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcatgtgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgctgacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaaatcctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674; Streptag II: 1681-1704; Linker sequences : 1540-1551, 1633-1638,1675-1680; P102A (naturally-occurring substitution): 304-306; I379V (naturally-occurringsubstitution): 1135-1137; M447V (naturally-occurring substitution): 1339-1341; T54H:160-162; T1030: 307-309; 11480: 442-444; S190I: 568-570; V2961: 886-888; D486S:1456-1458]SEQ ID NO: 13: Nucleotide Sequence Nucleotide Sequence Encoding Pre-coursor Polypeptideof pXCS852:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccactgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacatccaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539(only including native RSVF sequence); F2: 76-327; foldon: 1552-1632; Thrombin recognitionsequence: 1639-1656; His-tag: 1657-1674; Streptag II: 1681-1704; Linker sequences:1540-1551, 1633-1638, 1675-1680; P102A (naturally-occurring substitution): 304-306; I379V(naturally-occurring substitution): 1135-1137; M447V (naturally-occurring substitution):1339-1341; T54H: 160-162; S55C: 163-165; L188C: 562-564; D486S: 1456-1458]SEQ ID NO: 14: Nucleotide Sequence Encoding Pre-cursor Polypeptide of pXCS853:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtacacctgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant components (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; S55C: 163-165; L188C:562-564; S190I: 568-570; D486S: 1456-1458]SEQ ID NO: 15: Nucleotide Sequence Encoding Pre-cursor Polypeptide of pXCS855:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccactgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant features (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539;F2: 76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; T54H: 160-162; S55C:163-165; L188C: 562-564; S190I: 568-570; D486S: 1456-1458]SEQ ID NO: 16: Nucleotide Sequence Encoding Precursor Polypeptide of pXCS874:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtacaccagcgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtgtaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgctgacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtgcattatcaaggaggaagtgctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttcctccgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant features (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539; F2:76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; S155C: 463-465; S190I:568-570; S290C: 868-870; D486S: 1456-1458]SEQ ID NO: 17: Nucleotide Sequence Encoding Precursor Polypeptide of pXCS881:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccactgtgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgtgtggcgtgggctccgcaatcgcatccggagtggccgtgtccaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgtgtacatccaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtcaattatcaaggaggaaatcctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgtgtagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttccagccagttcagtgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant features (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539; F2:76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; T54H: 160-162; S55C:163-165; L1420: 424-426; L188C: 562-564; V2961: 886-888; N3710: 1111-1113; D486S: 1456-1458;E487Q: 1459-1461; D4895: 1465-1467]SEQ ID NO: 18: Nucleotide Sequence Encoding Precursor Polypeptide of pXCS898:atggaacttctgatcctgaaagccaacgcgattaccactatcctgactgccgtcaccttctgcttcgcatcgggacagaacattaccgaggagttctaccagtccacctgttcggcggtgtccaagggttacctctcggccctgagaactggctggtaccacagcgtgattactatcgagctgagcaacatcaaggagaacaagtgcaatggaacggacgcgaaggtcaagctgattaagcaggaactcgataagtacaagaacgccgtgaccgagctccagctgctgatgcaatcgacccctgccactaacaacagagctcgccgggaactgccgcgcttcatgaattacaccctcaacaacgcgaagaaaaccaacgtgaccctgtccaagaagcgcaagcggaggttcctgggattcctgttgggcgtgggctccgcaatcgcatccggagtggccgtgtgtaaagtgctgcatctggagggggaagtgaacaagatcaagtccgccctcctgtcaactaataaggcggtggtgtccctgagcaacggagtcagcgtgctgacaatcaaggtcctggacctcaagaactacatcgacaagcagctgttgcccatcgtcaacaagcagtcatgctcgattagcaatatcgaaaccgtgattgagttccagcagaagaacaacagactgctcgaaattacccgggagttttccgtgaacgccggagtgaccactcctgtgtccacctacatgcttacgaactccgaactgctcagcctcatcaacgatatgccgatcactaacgaccagaagaagttgatgagcaacaatgtgcagatcgtgcgccaacagtcctactcaatcatgtgcattatcaaggaggaaatcctcgcctatgtggtgcaattgcctctgtacggagtcatcgacacaccctgctggaagctgcacactagcccactctgtacgaccaacaccaaggaaggttccaacatctgcctgactaggaccgatcggggctggtattgcgataatgctgggtccgtgagcttcttcccgcaagccgagacttgcaaagtgcagtcaaaccgcgtgttctgtgacaccatgaacagcctgaccctgccatccgaagtcaacctctgcaacgtggacatctttaacccgaaatacgactgcaagattatgacctccaagaccgacgtcagcagctctgtcatcactagcctgggagctattgtgtcctgctacggaaagaccaaatgcactgcctcgaacaagaacagaggcatcatcaagaccttcagcaacggctgtgactacgtgtccaacaagggagtggacaccgtgtccgtcgggaacaccctgtactacgtgaacaagcaggaggggaagtcgctctacgtcaagggggaaccgattatcaatttctacgaccccctggtgttcccttccgacgagttcgatgcctccatatcccaagtcaacgagaagatcaaccagtctcttgccttcatccggaagtcggacgaactgctgtccgccatcggtggctatattccggaagcccccagggatggacaggcctacgtgcggaaggatggagaatgggtgcttttgtccaccttcctgggcggtctggtgccccgcggctcacaccatcatcaccaccacggttcgtggtcccaccctcaatttgagaagtga[Relevant features (bp coordinates): Signal sequence: 1-75; pep27: 328-408; F1: 409-1539; F2:76-327; foldon: 1552-1632; Thrombin recognition sequence: 1639-1656; His-tag: 1657-1674;Streptag II: 1681-1704; Linker sequences: 1540-1551, 1633-1638, 1675-1680; P102A(naturally-occurring substitution): 304-306; I379V (naturally-occurring substitution):1135-1137; M447V (naturally-occurring substitution): 1339-1341; T54H: 160-162; S155C:463-465; S190I: 568-570; S290C: 868-870; V296I: 886-888]SEQ ID NO: 19: Amino Acid Sequence of Precursor Polypeptide of pXCS847:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGVVYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPACNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; P102A (naturally-occurring substitution); I379V (naturally-occurring substitution);M447V (naturally-occurring substitution); T103C, I148C, S190I, D486S]SEQ ID NO: 20: Amino Acid Sequence of Precursor Polypeptide of pXCS851:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPACNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSSNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEKfRelevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; P102A (naturally-occurring substitution); I379V (naturally-occurring substitution);M447V (naturally-occurring substitution); T54H, T103C, I148C, S190I, V2961, D486S]SEQ ID NO: 21: Amino Acid Sequence of Precursor Polypeptide of pXCS852:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513 (only includingnative RSVF sequence); F2: 26-109; foldon: 518-544; Thrombin recognition sequence: 547-552;His-tag: 553-558; Streptag II: 561-568; P102A (naturally-occurring substitution); I379V(naturally-occurring substitution); M447V (naturally-occurring substitution); T54H(introduced mutation); S55C (introduced mutation); L188C (introduced mutation); D486S(introduced mutation)]SEQ ID NO: 22: Amino Acid Sequence of Heavy Chain Variable Domain ofAntibody D25:QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTVSSSEQ ID NO: 23: Amino Acid Sequence of Light Chain Variable Domain of Antibody D25:DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNVVYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKRSEQ ID NO: 24: Amino Acid Sequence of Heavy Chain Variable Domain of Antibody AM14:EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHVVVRQAPGKGLEVVVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSSSEQ ID NO: 25: Amino Acid Sequence of Light Chain Variable Domain of Antibody AM14:DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTVSEQ ID NO: 26: Amino Acid Sequence of Heavy Chain Variable Domain of Antibody AM22QVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHVVVRQAPGKGLEWMGGYEGEVDEIFYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGVTVTEAGLGIDDYWGQGTLVTVSSSEQ ID NO: 27: Amino Acid Sequence of Light Chain Variable Domain of Antibody AM22EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAVVYQQKPGQAPRLLIFGASSRATGIPVRFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSSIFTFGPGTKVDFKSEQ ID NO: 28: Amino Acid Sequence of Heavy Chain Variable Domain of Antibody MPE8:EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEVVVSSISASSSYSDYADSAKGRFTISRDNAKTSLFLQMNSLRAEDTAIYFCARARATGYSSITPYFDIWGQGTLVTVSSSEQ ID NO: 29: Amino Acid Sequence of Light Chain Variable Domain of Antibody MPE8:QS\NTQTPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNNRPSGVPDRFSASKSGTSASLAITGLQAEDEADYYCQSYDRNLSGVFGTGTKVTVLSEQ ID NO: 30: Amino Acid Sequence of Heavy Chain Variable Domain of Antibody 101F:QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARLYGFTYGFAYWGQGTLVTVSASEQ ID NO: 31: Amino Acid Sequence of Light Chain Variable Domain of Antibody 101F:DIVLTQSPASLAVSLGQRATIFCRASQSVDYNGISYMHWFQQKPGQPPKLLIYAASNPESGIPARFTGSGSGTDFTLNIHPVEEEDAATYYCQQIIEDPWTFGGGTKLEIKSEQ ID NO: 32: Amino Acid Sequence of Precursor Polypeptide of pXCS738:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLCGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMCSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137; F2: 26-109; foldon:518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II: 561-568;Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurring substitution); I379V(naturally-occurring substitution); M447V (naturally-occurring substitution); T54H(introduced mutation); S55C (introduced mutation); L142C (introduced mutation); L188C(introduced mutation); V296I (introduced mutation); N3710 (introduced mutation)]SEQ ID NO: 33 : Amino Acid Sequence of Precursor Polypeptide of pXCS780:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally- occurringsubstitution); S55C (introduced mutation); L188C (introduced mutation); D486S (introducedmutation)]SEQ ID NO: 34: Amino Acid Sequence of Precursor Polypeptide of pXCS830:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally-occurringsubstitution); T54H (introduced mutation); S55C (introduced mutation); L188C (introducedmutation); S190I (introduced mutation)]SEQ ID NO: 35: Amino Acid Sequence of Precursor Polypeptide of pXCS853:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally-occurring substitution); S55C (introduced mutation); L188C (introduced mutation);S190I (introduced mutation); D486S (introduced mutation)]SEQ ID NO: 36: Amino Acid Sequence of Precursor Polypeptide of pXCS855:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally-occurringsubstitution); T54H(introduced mutation); S55C (introduced mutation); L188C (introducedmutation); S190I (introduced mutation); D486S (introduced mutation)]SEQ ID NO: 37: Amino Acid Sequence of Precursor Polypeptide of pXCS874:MELLILKANAITTILTAVTFCFASGQN1TEEFYQSTCSAVSKGYLSALRTGVVYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally-occurringsubstitution); S155C (introduced mutation); S190I (introduced mutation); S290C (introducedmutation); D486S(introduced mutation)]SEQ ID NO: 38: Amino Acid Sequence of Precursor Polypeptide of pXCS881:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLCGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMCSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSQFSASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally-occurringsubstitution); T54H(introduced mutation); S55C (introduced mutation); L142C (introducedmutation); L188C (introduced mutation); V296I (introduced mutation); N3710 (introducedmutation); D486S (introduced mutation); E487Q(introduced mutation); D4895 (introducedmutation)]SEQ ID NO: 39: Amino Acid Sequence of Precursor Polypeptide of pXCS898:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK[Relevant features (amino acid residue coordinates): Signal sequence (not present in finalproduct): 1-25; pep27 (not present in final product): 110-136; F1: 137-513; F2: 26-109;foldon: 518-544; Thrombin recognition sequence: 547-552; His-tag: 553-558; Streptag II:561-568; Linker sequences: 514-517, 545-546, 559-560; P102A (naturally-occurringsubstitution); I379V (naturally-occurring substitution); M447V (naturally-occurringsubstitution); T54H(introduced mutation); S155C (introduced mutation); S190I (introducedmutation); S290C (introduced mutation); V296I (introduced mutation)]SEQ ID NO: 40: Amino acid Sequence of the T4 Fibritin Foldon:GYIPEAPRDGQAYVRKDGEWVLLSTFLSEQ ID NO: 271. Amino Acid Sequence of Precursor Polypeptide of pXCS899MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLGGLVPRGSHHHHHHGSWSHPQFEKSEQ ID NO: 272. Amino Acid Sequence of Precursor Polypeptide of pXCS1106MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIR

DELLHNVNAGKSTTNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGHHHHHHGSWSHPQFEKSEQ ID NO: 273. Amino Acid Sequence of Precursor Polypeptide of pXCS1107MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLH

NAGKSTTNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGHHHHHHGSWSHPQFEKSEQ ID NO: 274. Amino Acid Sequence of Precursor Polypeptide of pXCS1108MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKS

NIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGHHHHHHGSWSHPQFEKSEQ ID NO: 275. Amino Acid Sequence of Precursor Polypeptide of pXCS1109MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIR

DELLH

NAGKSTTNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGHHHHHHGSWSHPQFEKSEQ ID NO: 276. Amino Acid Sequence of Precursor Polypeptide of pXCS1110MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIR

DELLHNVNAGKS

NIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGHHHHHHGSWSHPQFEKSEQ ID NO: 277. Amino Acid Sequence of Precursor Polypeptide of pXCS1111MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLH

NAGKS

NIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEVVVLLSTFLGGHHHHHHGSWSHPQFEKSEQ ID NO: 278. Amino Acid Sequence of Precursor Polypeptide of pXCS1112MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGVVYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIR

DELLH

NAGKS

NIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

The invention claimed is:
 1. A mutant of a wild-type respiratorysyncytial virus (RSV) F protein, which mutant comprises a F1 polypeptideand a F2 polypeptide, wherein the mutant comprises at least oneintroduced amino acid mutation relative to the amino acid sequence ofthe wild-type RSV F protein, and wherein the introduced amino acidmutation comprises: (i) the pair of cysteine mutations 155C and 290C;and (ii) one or more cavity filling mutations selected from the groupconsisting of: 1) substitution of the amino acid at position 190 with I;2) substitution of the amino acid at position 54 with I, Y, L, H, or M;3) substitution of the amino acid at position 296 with I, Y, H, andwherein amino acid positions are numbered according to SEQ ID NO:1. 2.The mutant according to claim 1, wherein the cavity filling mutation isselected from the group consisting of: (1) substitution of the aminoacid at position 190 with I; (2) substitution of the amino acid atposition 54 with H; and (3) substitution of the amino acid at position296 with I.
 3. The mutant according to claim 1, which is in the form ofa trimer.
 4. The mutant according to claim 1, which has increasedstability as compared with the corresponding wild-type RSV F protein,wherein the stability is measured by binding of the mutant with antibodyAM14.
 5. The mutant according to claim 1, wherein the wild-type RSV issubtype A or subtype B.
 6. The mutant according to claim 2, wherein thecavity filing mutation is selected from the group consisting of: 54H,190I, and 296I.
 7. The mutant according to claim 1, further comprisingan electrostatic mutation.
 8. The mutant according to claim 7, whereinthe electrostatic mutation is selected from the group consisting of: (1)substitution of the amino acid at position 82, 92, or 487 by D, F, Q, T,S, L, or H; (2) substitution of the amino acid at position 315, 394, or399 by F, M, R, S, L, I, Q, or T; (3) substitution of the amino acid atposition 392, 486, or 489 by H, S, N, T, or P; and (4) Substitution ofthe amino acid at position 106 or 339 by F, Q, N, or W.
 9. A mutant of awild-type RSV F protein, which mutant comprises a F1 polypeptide and aF2 polypeptide, wherein the mutant comprises at least one introducedamino acid mutation relative to the amino acid sequence of the wild-typeRSV F protein, and wherein the introduced amino acid mutation comprises:(i) the pair of cysteine mutations 155C and 290C; (ii) an cavity fillingmutation; and (iii) an electrostatic mutation, wherein the cavityfilling mutation is selected from the group consisting of: (1)substitution of the amino acid at position 62 with I, Y, L, H, or M; (2)substitution of the amino acid at position 190 with I; (3) substitutionof the amino acid at position 54, 58, 189, 219, or 397 with I, Y, L, H,or M; (4) substitution of the amino acid at position 151 with A or H;(5) substitution of the amino acid at position 147 or 298 with I, L, H,or M; and (6) substitution of the amino acid at position 164, 187, 192,207, 220, 296, 300, or 495 with I, Y, H, wherein the electrostaticmutation is 486S, and wherein the amino acid positions are numberedaccording to SEQ ID NO:1.
 10. The mutant according to claim 1, whereinthe mutant comprises a combination of amino acid mutations selected fromthe group consisting of: (1) combination of 155C, 290C, and 54H; (2)combination of 155C, 290C, 296I; (3) combination of 155C, 290C, 54H, and296Y; (4) combination of 155C, 290C, and 190I; (5) combination of 155C,290C, 54H, and 190I; (6) combination of 155C, 290C, 54H, 496S; (7)combination of 155C, 290C, 190I, and 486S; (7) combination of 155C,290C, 296I; and 486S; (9) combination of 155C, 290C, 54H, 190I; and486S; (10) combination of 155C, 290C, 54H, 296I, and 486S (11)combination of 155C, 290C, 190I, 296I, and 485S; (12) combination of155C, 290C, 54H, 190I, 296I, and 486S; (13) combination of 155C, 290C,190I, and 296I; and (14) combination of 155C, 290C, 54H, 190I, and 296I.11. A mutant of a wild-type RSV F protein, which mutant comprises a F1polypeptide and a F2 polypeptide, wherein the mutant comprises at leastone introduced amino acid mutation relative to the amino acid sequenceof the wild-type RSV F protein, and wherein the introduced amino acidmutation comprises: (i) the pair of cysteine mutations 155C and 290C;(ii) at least one cavity filling mutation; and (iii) at least one pairof cysteine mutations in the HRB region, wherein the cavity fillingmutation is selected from the group consisting of: (1) substitution ofthe amino acid at position 62 with I, Y, L, H, or M; (2) substitution ofthe amino acid at position 190 with I; (3) substitution of the aminoacid at position 54, 58, 189, 219, or 397 with I, Y, L, H, or M; (4)substitution of the amino acid at position 151 with A or H; (5)substitution of the amino acid at position 147 or 298 with I, L, H, orM; and (6) substitution of the amino acid at position 164, 187, 192,207, 220, 296, 300, or 495 with I, Y, H, wherein the at least one pairof cysteine mutations in the HRB region is selected from the groupconsisting of: (1) 508C and 509C; (2) 515C and 516C; and (3) 522C and523C, and wherein the amino acid positions are numbered according to SEQID NO:1.
 12. A pharmaceutical composition comprising (i) an RSV Fprotein mutant according to claim 1 and (ii) a pharmaceuticallyacceptable carrier.
 13. The pharmaceutical composition according toclaim 12, wherein the F1 polypeptide and F2 polypeptide are from the Fprotein of RSV subtype B.
 14. The pharmaceutical composition accordingto claim 12, wherein the F1 polypeptide and F2 polypeptide are from theF protein of RSV subtype A.
 15. The pharmaceutical composition accordingto claim 14, further comprising a second mutant according to claim 1,wherein the F1 polypeptide and F2 polypeptide of the second mutant arefrom the F protein of RSV subtype B.
 16. The pharmaceutical compositionaccording to claim 12, which is a vaccine.
 17. A nucleic acid moleculecomprising a nucleotide sequence that encodes an amino acid sequence ofan RSV F protein mutant according to claim
 1. 18. A method of preventingRSV infection in a subject, comprising administering to the subject aneffective amount of the pharmaceutical composition according to claim12.
 19. A pharmaceutical composition comprising (i) an RSV F proteinmutant according to claim 9 and (ii) a pharmaceutically acceptablecarrier.
 20. A method of preventing RSV infection in a subject,comprising administering to the subject an effective amount of thepharmaceutical composition according to claim 19.